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Analysis of amyloid fibrils by site-directed spin labeling and electron paramagnetic resonance

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Analysis of amyloid fibrils by site-directed spin labeling and electron paramagnetic resonance

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ANALYSIS OF AMYLOID FIBRILS BY SITE-DIRECTED SPIN LABELING AND
ELECTRON PARAMAGNETIC RESONANCE

by

Min Chen

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

August 2009

Copyright 2009 Min Chen

ii
Acknowledgements

I would very much like to thank to professors, colleagues, friends and
family who have supported me throughout my Ph.D. studies.
First and foremost my thanks go to my supervisors, Professors Ralf
Langen and Jeannie Chen, for their guidance and continuing support during my
studies.
I want to thank Professor Sheena Radford, Drs. David Smith and Carol
Ladner (Univ. of Leeds, UK), for their collaborative effort on ß-2-microglobulin
project.
I would like to express my sincere thanks to the fellow researchers Drs.
Ani Der-Sarkissian, Peng Wu, Torsten Fisher, Balachandra Hegde and Mario
Isas, Ms. Christine Jao and Prabha Hegde, as well as Ms. Diana Gegala, who
have been kind enough to advise and help in their respective roles.
I am very grateful to The Charles and Charlotte Krown Fellowship from the
School of Pharmacy at USC, who partially funded my study.
The scientific findings of Chapter 3 were published on Journal of
Biological Chemistry in 2007, and copyright was owned by American Society for
Biochemistry and Molecular Biology, Inc.
Last, but not the least, I would like to dedicate this thesis to my family,
especially my parents, I want them to know that I am very grateful for their
unreserved love and encouragements throughout my studies, to which have
been a source of inspiration and moral support. Without their love and patience, I
would not have become how I am now. Thank you all!

iii
Table of Contents
Acknowledgements…………………………………………………………….ii

List of Tables……………………………………………………………………v

List of Figures…………………………………………………………………..vi

Abbreviations………………………………………………………………….viii

Abstract………………………………………………………………………….ix

Chapter 1: α-Synuclein Fibrils, ß-2-Microglobulin Fibrils and Related
Diseases……………………………………………………………………… ..1

1.1 General Properties of Amyloid Fibrils ..........................................1
1.2 α-Synuclein and Parkinson’s Disease .........................................3
1.3 ß-2-Microglobulin and Dialysis-related Amyloidosis ....................6
1.4 Chapter 1 References .................................................................9

Chapter 2: Site-Directed Spin Labeling and Electron Paramagenetic
Resonance…………………………………………………………………….16

2.1 Introduction................................................................................16
2.2 Mobility ......................................................................................17
2.3 Spin Exchange ..........................................................................20
2.4 Accessibility...............................................................................21
2.5 Distance Measurement..............................................................23
2.6 Chapter 2 References ...............................................................25

Chapter 3: Investigation of α-Synuclein Fibril Structure by Site-Directed
Spin Labeling………………………………………………………………….28

3.1 Abstract .....................................................................................28
3.2 Introduction................................................................................29
3.3 Experimental Procedures ..........................................................31
3.4 Results ......................................................................................35
3.5 Discussion.................................................................................53
3.6 Chapter 3 References ...............................................................59

Chapter 4: Distance Measurement of α-Synuclein Fibril by Site-Directed
Spin Labeling………………………………………………………………….66

4.1 Abstract .....................................................................................66
4.2 Experimental Procedures ..........................................................67
4.3 Results and Discussions ...........................................................71

iv
4.4 Chapter 4 References ...............................................................80

Chapter 5: Investigation of ß-2-Microglobulin Fibril Structure by Site-
Directed Spin Labeling and Electron Paramagnetic Resonance………..82

5.1 Abstract .....................................................................................82
5.2 Introduction................................................................................83
5.3 Experimental Procedures ..........................................................86
5.4 Results and Discussions ...........................................................90
5.5 Chapter 5 References .............................................................103

Chapter 6: Characterization of Spin Exchange Effect Using A Model
Protein D-Amphiphysin……………………………………………………..106

6.1 Abstract ...................................................................................106
6.2 Introduction..............................................................................106
6.3 Experimental Procedures ........................................................107
6.4 Results and Discussions .........................................................110
6.5 Chapter 6 References .............................................................116

Bibliography………………………………………………………………….117

v
List of Tables

Tabel 4-1. Summary of available α-synuclein double-cysteine mutants .76

Table 5-1. Comparison of ß2m structural features in M
H
ß2m crystal form,
solution and LS fibrils. ............................................................................92

Table 5-2. Comparison of EPR spectra central line width (C.L.W.)
between α-synuclein and ß2m................................................................97

vi
List of Figures

Figure 2- 1. Chemical structure of the paramagnetic side chain R1 and its
diamagnetic analogue, R1'. ......................................................................... 17

Figure 2- 2. Illustration of central line width and amplitude in EPR
spectrum...................................................................................................... 19

Figure 2- 3. Normalized EPR spectrum of R1-labeled 115ter α-synuclein in
solution. ....................................................................................................... 19

Figure 2- 4. Normalized EPR spectrum of R1-labeled ß-2-microglobulin at
position 81 in Long-straight fibrils.........................................................................21

Figure 3- 1. Chemical structure of the paramagnetic side chain R1 and its
diamagnetic analogue, R1’. ......................................................................... 34

Figure 3- 2. EPR spectra of R1-labelee α-synuclein (115ter) in soluble and
fibrillar forms................................................................................................ 38

Figure 3- 3. Spin-dilution experiments of α-synuclein (115ter) fibrils
containing varying mixtures of R1 and R1’ labels at position 52.................. 41

Figure 3- 4. Quantative analysis of dipolar spin-spin interaction at low
percentages of R1. ...................................................................................... 44

Figure 3- 5. EPR spectra of α-synuclein (115ter) fibrils containing single
labels at positions 34 – 101. ........................................................................ 47

Figure 3- 6. EPR spectra of α-synuclein (115ter) fibrils containing single
labels at selected sites in the N- and C- terminal regions............................ 48

Figure 3- 7.Graphic illustration of EPR data as a function of residue
number. ....................................................................................................... 50

Figure 3- 8. Schematic illustration of α-synuclein fibril structure
highlighting the parallel, in-registrer arrangement of multiple strands.......... 56

Figure 4- 1. Four-pulse DEER sequence. ...........................................................70
Figure 4- 2. Distance measurements in α-synuclein (115ter) fibrils from
continuous wave EPR spectroscopy. .......................................................... 72

Figure 4- 3. Schematic illustration of the experimental design to generate α-
synuclein double-cysteine mutants for potential distance measurements. .. 75

vii
Figure 5- 1. Morphological types of ß2m fibrils formed in vitro. ................... 85

Figure 5- 2. Chemical structure of the paramagnetic side chain R1. ........... 87

Figure 5- 3. Spectra of 100% R1-labeled ß2m monomers in pH 7, 25 mM
NaP and 30% sucrose................................................................................. 91

Figure 5- 4. Spectra of 100% R1-labeled ß2m LS fibrils (in pH 2.5, no salt).
.................................................................................................................... 95

Figure 5- 5. Comparison of spectra of 100% R1-labeled ß2m LS fibrils (in
pH 2.5, no salt) versus random precipitates. ............................................... 96

Figure 5- 6. Spin dilution of R1-labeled ß2m at position 61 in LS fibrillar
form. ............................................................................................................ 97

Figure 5- 7. Comparison of EPR Spectra derived from 100% R1-labeled α-
synuclein and ß2m LS fibrils........................................................................ 98

Figure 5- 8. Spectra of 25% R1-labeled ß2m LS fibrils (in pH 2.5, no salt).100

Figure 5- 9. Plot of mobility and oxygen accessibility of 25% R1-labeled
ß2m LS fibrils (in pH 2.5, no salt). ............................................................. 101

Figure 5- 10. EPR Spectra of 100% R1-labeled ß2m WL fibrils (in pH 2.5,
0.4 M salt).................................................................................................. 102

Figure 6- 1. Chemical structure of the paramagnetic side chain R1 and its
diamagnetic analogue, R1’. ....................................................................... 109

Figure 6- 2. A snapshot of D-amphiphysin dimer crystal structure. .......... 113

Figure 6- 3. EPR spectra of 100% R1-labeled D-amphiphysin measured
at room temperature and frozen state. ...................................................... 114

viii
Abbreviations
PD, Parkinson disease;
AD, Alzheimer disease;
NAC, non-amyloid ß component;
SDSL, site-directed spin labeling;
EPR, electron paramagnetic resonance;
ssNMR, solid-state nuclear magnetic resonance;
H/D exchange, hydrogen-deuterium exchange;
EM, electron microscopy;
CD, circular dichroism;
ß2m, ß-2-microglobulin;
DRA, dialysis related amyloidosis;
LS, long-straight;
WL, worm-like;
apoE, Apolipoprotein E;
GAGs, glycosaminoglycans;
PGs, proteoglycans;
N/A, not available.

ix
Abstract
This thesis discusses the application of site-directed spin labeling coupled
with electron paramagnetic resonance spectroscopy in the structural analysis of
amyloid fibrils derived from α-synuclein and ß-2-microglobulin.
The first part of study is focused on α-synuclein fibrils. We observe single-
line, exchange-narrowed EPR spectra for the majority sites located within the
core region of the fibrils. The core region is extended from aa 36 - 98 and tightly
packed in a parallel, in-register style, with same residues from different peptides
stacking on top of each other. Only few areas are significantly less ordered.
Oxygen accessibility data indicates the possible location of putative ß-sheet
structures. We successfully establish and validat the method to measure the
intra-molecular distances. And such intra-molecular distances obtained from cw
and pulsed EPR measurements confirm some of the tested regions indeed form
ß structures. Additional intra-molecular distances constraints to be generated
would help us eventually build an accurate three-dimensional model of α-
synuclein fibril.
The second part of research is focused on ß-2-microglobulin. The major
differences of these two studies lie in that ß-2-microglobulin has a native
structure in contrast to the largely disordered native α-synuclein; additionally, with
ß-2-microglobulin, different types of fibrils are produced, these makes this study
more complicated and more interesting. In this study, we focus our attention on
its secondary structural features in solution state; in both long-straight and worm-
like fibril forms. We notice significant conformational changes take place upon

x
monomers were transformed into fibrils. Surprisingly, long-straight and worm-like
fibrils, which own different external gross morphologies, do show distinct
structural features. Similar to α-synuclein fibrils, single-line, exchange-narrowed
spectra are observed in most of the tested sites located within the long-straight
fibrils core region, indicating a parallel, in-register packing signature. And the
terminuses of the protein are much less ordered. In contrast, with the worm-like
fibrils, no clear single-line; exchange-narrowed spectra are detected in any of the
sites we studied, suggesting that the worm-like fibrils pick up a different structure.
Finally, as the single-line, exchange-narrowed EPR spectra phenomenon
have been observed by both systems of α-synuclein fibrils and ß-2-microglobulin
long-straight fibrils, as well as shared by many other naturally occurring amyloid
fibrils, we have utilized a model protein D-amphiphysin to characterize the origin
of spin exchange. Our study have shown that in such simulated in vitro system,
4 spin labels in close contact (about 5 Å proximity) with each other are sufficient
to result in spin exchange.

1
CHAPTER 1:
α-SYNUCLEIN FIBRILS, ß-2-MICROGLOBULIN FIBRILS AND RELATED
DISEASES

1.1 General Properties of Amyloid Fibrils
Amyloid fibrils are abnormal, insoluble, relatively proteinase-resistant
structures, and have been found to be associated with more than 20 different
diseases. Such depositions are hallmarks of many neurodegenerative disorders.
In each of these diseases, one specific peptide or protein or protein fragment
alters from its native state into insoluble deposits. The protein misfolding is a
stepwise process involving a number of oligomeric species, and ultimately leads
to fibril formation and deposition, such as α-synuclein fibrils in Parkinson’s
Disease (PD) and Aß fibrils in Alzheimer’s Disease (AD). Those proteins known
to aggregate in vivo were found to be able to reproduce fibrillar aggregates in
vitro (Stefani et al. 2003).
Thus far, the underlying protein misfolding mechanism remains poorly
understood. It is crucial to decipher the structures of the misfolded forms to
better understand why and how it takes place and eventually develop effective
therapeutics to prevent it, particularly has been the structure of amyloid fibrils,
since they represent the endpoint of misfolding.
Although amyloid fibrils originate from peptides and proteins that might
lack similarity in size, sequence and/or native structure, they seem to share some
common characteristics both in external gross morphology and internal structure.
Commonalities include their ability to bind Congo Red and thioflavins (Sunde et
al. 1997), the presence of long, straight and unbranched classical fibrils under

2
electron microscope (EM) (Cohen et al. 1959), as well as containing “cross-ß”
structure indicated by X-ray fiber diffraction (Pauling et al. 1951; Eanes et al.
1968; Geddes et al. 1968; Sunde et al. 1998).
Resolving structures of amyloid fibrils has been problematic because of its
enormity and insolubility, conventional techniques such as X-ray crystallography
or solution state NMR cannot be applied. However, recent work has
demonstrated site-directed spin labeling (SDSL) together with electron
paramagnetic resonance (EPR) spectroscopy as a powerful technique
particularly for such studies, greatly due to the fact that that no crystallization is
required and it can be applied to soluble or insoluble proteins of any size.
Though little is known at present about the detailed three-dimensional structure
of any amyloid fibril, more structural and dynamic details of amyloid fibrils have
been acquired through EPR studies. Such EPR studies have indicated that fibrils
of A β
1-40
, α-synuclein, tau, IAPP share similar structural organization, that
individual ß-strands within their highly ordered core regions are arranged in a
parallel fashion. These data are consistent with that of Aß
10-35,
yeast prion
Ure2p
10-39
and ß-2-microglobulin fragment (S20-K41) structures studied by solid
state NMR (ssNMR) (Fraser et al. 1994; Benzinger et al. 1998; Antzutkin et al.
2000; Serag et al. 2001; Torok et al. 2002; Der-Sarkissian et al. 2003;
Jayasinghe et al. 2004; Margittai et al. 2004; Chan et al. 2005; Iwata et al. 2006),
suggesting that a common molecular fibril assembly mechanism is conserved
among many naturally occurring amyloid fibrils.

3
1.2 α-Synuclein and Parkinson’s Disease
Parkinson’s disease (PD) is the second most common neurodegenerative
disorder after Alzheimer’s disease (AD). Clinically PD is characterized by tremor,
rigidity and bradykinesia. Pathologically PD is characterized by loss of
dopaminergic neurons in substantia nigra and the presence of Lewy bodies (LBs)
and Lewy neuritis (LNs) in the brain, in which α-synuclein fibrils constitute the
major components (Spillantini et al. 1997; Spillantini et al. 1998). With the
advanced age, the incidence of neurodegenerative diseases, such as AD and PD,
increases dramatically. Currently PD afflicts 1% of people over age of 60, more
than 1.5 million people in US (Forman et al. 2004). Due to the lack of effective
treatments, these diseases have posed significant medical, social and financial
burdens on the society.
α-Synuclein is a small (140aa), natively unfolded (Weinreb et al. 1996),
intracellular protein that is widely expressed in the nervous system, especially in
presynaptic nerve terminals (Maroteaux et al. 1988; Iwai et al. 1995). However,
its physiological functions have not been fully elucidated.
Initially human α-synuclein was identified as the precursor protein (NACP)
for the non-amyloid beta component (NAC) from AD amyloid plaques (Ueda et al.
1993). NAC is a hydrophobic core in the middle portion of α-synuclein (Weinreb
et al. 1996). C-terminal is a negatively charged tail, without it, the truncated
protein is prone to aggregate (Choi et al. 2002; Kim et al. 2002; Park et al. 2002;
Uversky et al. 2002).

4
Although the cause of almost all cases of PD remains unknown, a growing
body of evidences has implicated α-synuclein as a key protein participating in its
pathogenesis. For example, α-synuclein mutations including A53T
(Polymeropoulos et al. 1997), A30P (Kruger et al. 1998), E46K (Zarranz et al.
2004), as well as duplication and triplication of the wild-type gene (Singleton et al.
2003; Chartier-Harlin et al. 2004; Ibanez et al. 2004), all could give rise to rare
familial PD. Furthermore, the aggregated form of α-synuclein known as amyloid
fibrils was identified as the major component of LBs or LNs (Spillantini et al. 1997;
Spillantini et al. 1998), indicating that it accumulates at high levels in sporadic PD.
Besides, its abnormal deposition is not limited in PD (Giasson et al. 2000;
Jellinger 2003), but also in AD (Jellinger 2003), dementia with Lewy bodies (DLB)
(Jellinger 2003), and multiple system atrophy (MSA) (Duda et al. 2000).
Additionally, its selective cellular damage to dopaminergic neurons has been
demonstrated in cell culture studies (Xu et al. 2002). Moreover, animal models of
human α-synuclein developed in Drosophila (Feany et al. 2000), mice (Masliah et
al. 2000; Lee et al. 2002), rats (Kirik et al. 2002) and marmoset monkeys (Smith
et al. 2001; Kirik et al. 2003) support its causative role in disease. Despite these,
the underlying mechanism of misfolding and aggregation of α-synuclein still is
unclear.
Over the last decade, numerous efforts have been made to capture the
structural features of α-synuclein fibrils. Under EM, fibrils are typically 6-10 nm
width with variable length, straight and/or twisted, and consist of two to six
“protofilaments”, each about 2 nm diameter (Rochet et al. 2000). Circular

5
Dichroism (CD) study indicated the presence of predominant β-sheet structure in
fibrils (Serpell et al. 2000). X-ray diffraction showed the cross- β structure, with β-
strands running perpendicular to the fiber axis, backbone hydrogen bonds
forming parallel to the fiber axis, and with a main chain separation around 4.7-
4.8Å, a inter-sheet separation around 10 Å (Serpell et al. 2000). As for its fibril
domain organization, EPR study from our lab indicates fibril forming core is
flanked by a less ordered N-terminal and an unfolded C-terminal (Der-Sarkissian
et al. 2003), which was confirmed by hydrogen-deuterium (H/D) exchange,
ssNMR studies recently (Del Mar et al. 2005; Heise et al. 2005). And the fibril
forming core seems much more extended than NAC region. Similar results with
minor deviation have been shown, including proteinase K digestion resistance
segment of residues 31-109 (Miake et al. 2002); EPR core extending from
residue 34-101; H/D exchange protecting region of residues 39-101; and ssNMR
core composed of at least residues 38-95. Moreover, EPR study suggests fibril
strands take up parallel, in-register arrangements, which are also observed in
fibrils of Aß
10-35
, A β
1-40
, tau, IAPP and yeast prion Ure2p
10-39
(Fraser et al. 1994;
Benzinger et al. 1998; Antzutkin et al. 2000; Torok et al. 2002; Jayasinghe et al.
2004; Margittai et al. 2004; Chan et al. 2005).
Beyond these valuable insights, however, these studies are not complete
and not conclusive yet. More evidence needs to resolve the whole picture of α-
synuclein fibril structure.

6
1.3 ß-2-Microglobulin and Dialysis-related Amyloidosis
Dialysis-related amyloidosis (DRA) is a serious yet increasingly common
complication that is associated with patients undergoing long-term dialysis. In
DRA, ß-2-microglobulin (ß2m) has been found to be the major player in the
amyloid fibrils that mainly deposit in osteoarticular joint tissues and cause various
complications such as carpal tunnel syndrome, destructive spondyloarthropathy,
bone cysts, and various organ dysfunctions (Gejyo et al. 1985; Shirahama et al.
1985). The incidence of DRA goes up steadily with the length of survival. As
such, the prevalence of carpal tunnel syndrome rises with years of dialysis that
up to 50% of patients had developed this complication after 20 years affected
and even higher percentage after 25 years (Gejyo et al. 2003). Prevention of
onset or progression; symptomatic therapy such as conservative treatment,
physical therapy and orthopedic procedures; and renal transplantation have been
generally used in the amyloidosis intervention, however, more effective
treatments are still lacking (Gejyo et al. 2003)
ß-2-microglobulin (ß2m) is the non-covalently bound light chain of the
class I human leukocyte antigen (HLA class I) and it has a central role in cellular
immunology ensuring MHC-1 (the class I major histocompatibility complex) folds
and assemblies correctly. And ß2m is degraded and excreted in the kidney. In
DRA, once ß2m can not be metabolized correctly and efficiently, free ß2m
accumulates up to 60 folds higher than normal level. Such elevated serum levels
of ß2m are the main cause for the onset of amyloidosis. Excess ß2m is a
necessity to start the process, but it is probably not sufficient in itself, because,

7
unfortunately, removal of ß2m does not obviously help reduce the amyloid
deposits after long-term dialysis (Gejyo et al. 2003).
ß2m is a 99-residue monomeric protein that contains 2 native cysteines
forming a disulfide bond to stabilize the protein. Native ß2m displays a seven ß-
strands ß-sandwich structure. Because of its relative smaller size, unique native
structure and its clinical significance to DRA, extensive efforts had been made
toward understanding the protein misfolding mechanism (Chatani et al. 2005).
In the initial in vitro studies, ß2m amyloid fibrils were produced at low pH
(2.0 – 3.0). Though it was rarely known how the precursors were populated from
the native proteins at the physiological condition. The in vitro studies (Gejyo et al.
2003) indicated that various specific amyloid-associated molecules that affect
ß2m conformation and stability, such as apolipoprotein E (apoE),
glycosaminoglycans (GAGs), and proteoglycans (PGs), could play a significant
role in stabilizing the formation and deposition of amyloid fibrils at neutral pH
(Naiki et al. 2005). It is noted that, in vitro, the precursor formed at pH 2.5 is
more highly unfolded. And such partial unfolding of this native protein is believed
to be the prerequisite for fibril assembly, though the precise mechanism remains
unknown (Yamamoto et al. 2007). Under acidic condition, different types of fibrils
are generated that are differ in their external gross morphologies, including long-
straight (LS) fibrils that have several protofilaments twisted in a left-handed
helical way; flexible and nodular worm-like (WL) fibrils; and the much shorter rod-
like (RL) fibrils formed under moderate ionic-strength buffer but no salt (Radford
et al. 2005).

8
Though the atomic structure of the ß2m fibrils is still not available yet,
various biophysical approaches have been utilized to obtain the secondary
structure features of the ß2m fibrils. These methodologies include: X-ray fiber
diffraction (Smith et al. 2003); AFM (Kad et al. 2003; Gosal et al. 2006); H/D
exchange (Hoshino et al. 2002; Yamaguchi et al. 2004); solution and solid state
NMR (Iwata et al. 2006); pepsin limited proteolysis and ESI-MS (Myers et al.
2006); proline scanning mutagenesis (Chiba et al. 2003) and trypophan
mutagenesis (Kihara et al. 2006) and etc. Overall, these studies have suggested
that the terminuses of the fibrils were less ordered, the core region was located in
the middle stretch, though the beginning and the end of the cores slightly varied
from each other depending on which method was utilized. The most recent
ssNMR study of the K3 peptide (S20-K41) indicated that the K3 peptide formed
fibrils similar to full-length LS fibrils, and the each K3 molecule was stacked in a
parallel and staggered fashion. Nevertheless, the site-specific structural
information of the full length fibrils is lacking, neither is known about the structural
difference between the different morphology types of fibrils.

9
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16
CHAPTER 2:
SITE-DIRECTED SPIN LABELING AND ELECTRON PARAMAGENETIC
RESONANCE

2.1 Introduction
Site-directed spin labeling (SDSL) together with electron paramagnetic
resonance (EPR) has become a powerful technique particularly for use in such
studies to elucidate the structure and dynamics of membrane proteins and
amyloid fibrils, owing to its advantages that no crystallization is required and it
can be applied to soluble or insoluble proteins of any size. (Serag et al. 2001;
Torok et al. 2002; Der-Sarkissian et al. 2003; Jao et al. 2004; Jayasinghe et al.
2004; Margittai et al. 2004; Margittai et al. 2006; Margittai et al. 2006; Chen et al.
2007; Apostolidou et al. 2008; Jao et al. 2008; Margittai et al. 2008).
In SDSL, the amino acid residue of interest is substituted by a cysteine,
which subsequently reacts with paramagnetic nitroxide spin label, the most
commonly used [1-Oxyl-2,2,5,5-tetramethyl-D-pyrroline-3-methyl]-
methanethiosulfonate (MTSL), to generate side chain R1 (Figure 2-1). The
substitution of side chain R1 is well tolerated in various protein systems and has
little effect in disturbing the aim protein’s original backbone structures
(McHaourab et al. 1996; Hubbell et al. 1998). The introduced nitroxide serves as
a reporter to sense any changes in its local structural environment; this
information is captured by EPR spectroscopy.
Three key parameters are obtained via analyzing EPR spectra: mobility of
the side chain; solvent accessibility of the probe to environmental paramagnetic
reagents; and the distance between two paramagnetic centers (Hubbell et al.
2000). All are informative for secondary structure determination, i.e. to identify
tertiary contact sites, map the type and location of secondary structural elements
and measure specific inter-residue distances. Eventually, a collective
assessment of all of these geometric constraints will enable us to construct
accurate three-dimensional models (Jao et al. 2008).

Figure 2- 1. Chemical structure of the paramagnetic side chain R1 and its
diamagnetic analogue, R1'.
(Adapted from (Chen et al. 2007) ).

2.2 Mobility
Mobility information is obtained by measuring the inverse central line
width ( ΔH
0
-1
) of a EPR spectrum (Figure 2-2), and can be used to distinguish
exposed, buried, loop or tertiary contact sites, and monitor protein conformation
changes (McHaourab et al. 1996; Margittai et al. 2001; Isas et al. 2002).

17

18
Residue mobility varies from each other and primarily results from the
differences of tertiary interactions at local environment. Without constraint from
tertiary interactions, the spin label tumbles fast. The rapid tumbling of spin label
can average out the direction dependent anisotropic effect, and comes with a
spectrum with sharp line-shape and small central line width. For instance, an
unstructured site would present a characteristic three-sharp line spectrum (see
Figure 2-3). In contrast, for those sites that are buried and have rigid motion, the
presence of tertiary interactions would slow down the motion of spin label. And
anisotropic effect cannot be effectively averaged out; in return, the anisotropic
effect will contribute to the changes of EPR spectra line width. In response, the
respective EPR line-shape becomes broadened with outer two peaks separated
further more; the central line width gets bigger and amplitude get smaller.
It’s noted that strong spin-spin interaction (interaction between two
electrons) also causes line-shape broadening and signal amplitude reduction.
Therefore to exclusively analyze site-specific mobility information, spin-spin
interaction needs to be eliminated before subtracting any line-shape features.
For example diluting spin labels far enough with nonparamagnetic analog (see
Figure 2-1) or with unlabeled wild type could help reduce the spin-spin
interactions.
Such mobility analysis could be used to locate the precise boundary of the
fibril core region and determine the location of putative ß-strands and loop/turn
region within the fibril core region.

Amplitude
0 H Δ

Figure 2- 2. Illustration of central line width and amplitude in EPR spectrum.
Central Line Width ( ΔH
0
) describes the mobility of the spectrum that inverse
central line width directly correlates with the mobility. And the amplitude of the
central line describes the intensity of the EPR signal.

Figure 2- 3. Normalized EPR spectrum of R1-labeled 115ter α-synuclein in
solution.
(Adapted from (Chen et al. 2007) ).

19

20
2.3 Spin Exchange
As mentioned earlier, strong spin-spin interaction causes a broadened
line-shape and smaller signal amplitude for a given EPR spectrum. In an
extreme case, spin labels are close enough, the orbital of the paramagnetic
centers could overlap that would allow the two or more un-paired electrons to
freely exchange with each other on the EPR time scale. This is called spin
exchange (Molin et al. 1980), and it would result in a single-line exchange-
narrowed EPR spectrum, as shown in Figure 2-4.
The occurrence of spin exchange phenomenon with nitroxide labels have
been routinely observed in such systems as crystals of nitroxide labels, bi-/ tri-/
higher radicals, or rapidly colliding spin labels in solution/ micelles/ membranes
(Lajzerowicz-Bonneteau 1976; Molin et al. 1980; Hanson 1996). It was very
unique to detect the presence of spin exchange in the amyloid fibrils systems,
including Aß, Tau, α-synuclein, IAPP and etc. (Torok et al. 2002; Der-Sarkissian
et al. 2003; Jayasinghe et al. 2004; Margittai et al. 2004; Chen et al. 2007).
To produce the spin exchange, it was estimated that the distance upper
limit to be about 7 Å in a two-electron system and with even shorter distance
constraints for a multi-electron system (Margittai et al. 2008). However, it was
not clear in the aforementioned amyloid fibrils systems, exactly how many spin
labels are sufficient and exactly in what distance proximity to have contact with
each other.

Figure 2- 4. Normalized EPR spectrum of R1-labeled ß-2-microglobulin at
position 81 in Long-straight fibrils.

2.4 Accessibility
Solvent accessibility is determined from the collision frequency of the
nitroxide with paramagnetic reagents through power saturation analysis, and can
be used to explore side chain exposure to the environment.
An EPR spectrum is generated from the energy absorption between
different energy states. In power saturation, increasing the microwave power will
lead to the increase of the EPR signals as more electrons are excited, until it
reaches the stage that the two energy levels are equally distributed. At that point,
the further increasing of the microwave power will not further increase the EPR
signal outputs but reduce it, as most electrons have been excited and cannot
absorb any more energy. The P
1/2
is used to describe the effect of power
saturation, defined by the microwave power required saturating half of the

21

22
maximum signal amplitude. The presence of the paramagnetic reagents could
help excited electrons to transfer energy and come down to the initial state,
therefore elongate the process and increase the P
1/2
. The collision rate between
the spin probes and the paramagnetic reagents decides how effective the de-
saturation is. And because the increase of P
1/2
( ΔP
1/2
) is in proportion with the
collision rate, it has become a convenient index to assess the accessibility of the
spin label to the environment. The collision frequency is calculated from power
saturation curves, measured as EPR spectra amplitude changes as a function of
microwave power in the presence or absence of paramagnetic reagents
(Altenbach et al. 1994).
The commonly used reagents are molecular oxygen, which is hydrophobic
and exhibits preferential solubility in lipid membranes; and NiEDDA (Ni (II)
ethylenediamine diacetate), a neutral water soluble molecule, preferentially
partitions in aqueous environment. Therefore, solvent-exposed sites tend to
have a high accessibility to NiEDDA, lipid-exposed surface sites have a high
accessibility to oxygen, and sites in the protein interior have low accessibility to
both. For a regular secondary structure, as nitroxide scan through its sequence,
residues with similar accessibility are sampled out, reflected in periodic variation
of accessibility. For example trans-membrane α-helix has a periodicity of 3.6,
while ß-strand has a periodicity of 2 (McHaourab et al. 1996). Such oxygen
accessibility analysis could be applied to determine the location of putative ß-
strands in the amyloid fibrils.

23
2.5 Distance Measurement
Distance measurements are based on the distance dependence of dipole-
dipole coupling between two electrons. The magnetic interactions between two
unpaired electrons from different nitroxides (on the same or different molecules)
include exchange interaction, static dipolar interaction and modulation of the
dipolar interaction by the residual motion of the spin label side chains (Steinhoff
2004).
Exchange interaction requires orbital overlap of nitroxides from multiple
spin labels, hence acts in a short-range distance of less than 8 Å.
Static dipolar interaction is a longer-range effect that leads to reduction of
amplitude and broadening of continuous wave (cw) EPR spectra if distance is
less than 20 Å. Distances of 8 - 20 Å can be reliably determined through line
shape analysis of cw EPR spectra (Rabenstein et al. 1995; Steinhoff et al. 1997),
for example by fitting the spectral lineshape of doubly labeled mutant to the
lineshape of the sum of the single mutants convoluted against Pake pattern line
broadening function, as validated by X-ray data (McHaourab et al. 1997;
Altenbach et al. 2001).
Beyond 20 Å, spectral broadening caused by dipolar interaction is much
less than other homogeneous and inhomogeneous contributions since dipolar
interaction decays with the third power of inter-spin distance in a slow motion
regime; distance then can be estimated through modulation of the dipolar
interaction by pulsed EPR, which can reach distance of 15 – 80 Å (Lakshmi et al.
2001; Jeschke 2002; Milov et al. 2003; Jeschke et al. 2004; Hilger et al. 2005).

24
Through the combination use of cw and pulsed EPR, distance of 8 – 80 Å
could be examined and provides excellent conformational constraints for amyloid
fibrils and other protein systems. In fibrils system, such distance measurements
could be adopted in revealing the relative arrangement and orientations of
secondary structural elements with respect to each other.

25
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Biol Chem 276(16): 13169-77.

Margittai, M. and Langen, R. (2004). "Template-assisted filament growth by
parallel stacking of tau." Proc Natl Acad Sci U S A 101(28): 10278-83.

Margittai, M. and Langen, R. (2006). "Side Chain-dependent Stacking Modulates
Tau Filament Structure." J Biol Chem 281(49): 37820-7.

Margittai, M. and Langen, R. (2006). "Spin labeling analysis of amyloids and
other protein aggregates." Methods Enzymol 413: 122-39.

Margittai, M. and Langen, R. (2008). "Fibrils with parallel in-register structure
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paramagnetic resonance spectroscopy." Q Rev Biophys 41(3-4): 265-97.

27
McHaourab, H. S., Lietzow, M. A., Hideg, K. and Hubbell, W. L. (1996). "Motion
of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and
dynamics." Biochemistry 35(24): 7692-704.

McHaourab, H. S., Oh, K. J., Fang, C. J. and Hubbell, W. L. (1997).
"Conformation of T4 lysozyme in solution. Hinge-bending motion and the
substrate-induced conformational transition studied by site-directed spin
labeling." Biochemistry 36(2): 307-16.

Milov, A. D., Tsvetkov, Y. D., Formaggio, F., Oancea, S., Toniolo, C. and Raap, J.
(2003). "Aggregation of spin labeled trichogin GA IV dimers: distance distribution
between spin labels in frozen solutions by PELDOR data." J. Phys. Chem. B 107:
13179-13727.

Molin, Y. N., Salikhov, K. M. and Zamaraev, K. I. (1980). "Spin Exchange."
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Rabenstein, M. D. and Shin, Y. K. (1995). "Determination of the distance
between two spin labels attached to a macromolecule." Proc Natl Acad Sci U S A
92(18): 8239-43.

Serag, A. A., Altenbach, C., Gingery, M., Hubbell, W. L. and Yeates, T. O. (2001).
"Identification of a subunit interface in transthyretin amyloid fibrils: evidence for
self-assembly from oligomeric building blocks." Biochemistry 40(31): 9089-96.

Steinhoff, H. J. (2004). "Inter- and intra-molecular distances determined by EPR
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Steinhoff, H. J., Radzwill, N., Thevis, W., Lenz, V., Brandenburg, D., Antson, A.,
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fibrils studied by site-directed spin labeling." J Biol Chem 277(43): 40810-5.

28
CHAPTER 3:
INVESTIGATION OF α-SYNUCLEIN FIBRIL STRUCTURE BY SITE-
DIRECTED SPIN LABELING

3.1 Abstract

The misfolding and fibril formation of α-synuclein plays an important role in
neurodegenerative diseases such as Parkinson disease. Here we used electron
paramagnetic resonance spectroscopy, together with site-directed spin labeling,
to investigate the structural features of α-synuclein fibrils. We generated fibrils
from a total of 83 different spin-labeled derivatives and observed single-line,
exchange-narrowed EPR spectra for the majority of all sites located within the
core region of α-synuclein fibrils. Such exchange narrowing requires the orbital
overlap between multiple spin labels in close contact. The core region of α-
synuclein fibrils must therefore be arranged in a parallel, in-register structure
wherein same residues from different molecules are stacked on top of each other.
This parallel, in-register core region extends from residue 36 to residue 98 and is
tightly packed. Only a few sites within the core region, such as residues 62 to 67
located at the beginning of the NAC region, as well as the N- and C-terminal
regions outside the core region, are significantly less ordered. Together with the
accessibility measurements that suggest the location of potential ß-sheet regions
within the fibril, the data provide significant structural constraints for generating
three-dimensional models. Furthermore, the data support the emerging view that
parallel, in-register structure is a common feature shared by a number of
naturally occurring amyloid fibrils.

29
3.2 Introduction
α-Synuclein is a small, predominantly presynaptic protein that is widely
expressed throughout the nervous system (Maroteaux et al. 1988; Iwai et al.
1995). Although its physiological functions are not yet fully understood, there is
ample evidence that α-synuclein plays an important role in the pathogenesis of
several neurodegenerative diseases (Goedert 2001; Soto 2003; Norris et al.
2004). α-Synuclein is 140 amino acids in length. A fragment within α-synuclein,
referred to as NAC, was first identified in deposits derived from patients with
Alzheimer disease (Ueda et al. 1993). Subsequently, fibrillar deposits of α-
synuclein were found to be a main component of Lewy bodies, which represent a
hallmark of Parkinson disease (PD) (Giasson et al. 2000; Jellinger 2003) as well
as other neurodegenerative diseases (Duda et al. 2000; Jellinger 2003).
Importantly, α-synuclein missense mutations, such as A53T, A30P and E46K
(Polymeropoulos et al. 1997; Kruger et al. 1998; Zarranz et al. 2004), as well as
duplication and triplication of the wild-type gene in humans (Singleton et al. 2003;
Chartier-Harlin et al. 2004; Ibanez et al. 2004), can give rise to rare familial PD.
Moreover, animal models of human α-synuclein overexpressed in Drosophila
(Feany et al. 2000), mice (Masliah et al. 2000; Lee et al. 2002), rats (Kirik et al.
2002) and marmoset monkeys (Smith et al. 2001; Kirik et al. 2003) support its
causative role in diseases.
The toxicity of α-synuclein and that of other amyloidogenic proteins has
been linked to a misfolding process that involves a number of oligomeric species
and ultimately results in fibril formation (Caughey et al. 2003). While the precise

30
mechanisms of toxicity remain to be fully elucidated, it is clear that some of the
misfolded oligomeric species are highly cytotoxic. In order to better understand
and ultimately prevent the misfolding process, it is important to decipher the
underlying molecular mechanisms. Inasmuch as α-synuclein fibril formation
represents the endpoint of the misfolding process and is considered a hallmark of
the aforementioned diseases, the analysis of its fibril structure has received
significant attention. Fiber diffraction studies have shown that fibril formation
results in formation of the classical “cross-ß” structure (Serpell et al. 2000). In
this cross-ß structure, individual ß-strands are arranged perpendicular to the fibril
axis spaced at distances of 4.7 to 4.8 Å apart from each other. A number of
studies using electron paramagnetic resonance (EPR) spectroscopy, proteolysis,
hydrogen-deuterium (H/D) exchange and solid-state nuclear magnetic resonance
(ssNMR) spectroscopy (Miake et al. 2002; Der-Sarkissian et al. 2003; Del Mar et
al. 2005; Heise et al. 2005) have suggested that the cross-ß core region is
located in the central portion of α-synuclein. While this core region is highly
structured, the N- and C-terminal regions appear to be less ordered, with the C-
terminus being largely unfolded. Surprisingly, it was found that the borders of the
core region extend significantly beyond the NAC region (residues 61 to 95).
Various studies suggest that the core region begins somewhere in the range of
residues 31 to 39 and ends around residues 95 to 109 (Miake et al. 2002; Der-
Sarkissian et al. 2003; Del Mar et al. 2005; Heise et al. 2005). Despite these
results and the initial assignment of secondary structure for selected parts of the

31
core region by ssNMR (Heise et al. 2005), the precise extent of the core region
and its three-dimensional structure remains unknown.
In the present study, we applied EPR spectroscopy, together with site-
directed spin labeling (SDSL) (Hubbell et al. 1998; Hubbell et al. 2000; Margittai
et al. 2006), to further investigate the structure of α-synuclein fibrils. We
generated 83 different spin-labeled derivatives of α-synuclein, including a
nitroxide scan wherein each residue of α-synuclein (from 30 to 101) was replaced
by spin label R1 (Fig. 1), one amino acid at a time. EPR analysis of fibrils grown
from these derivatives was performed in order to (a) define the precise location of
the core region, (b) assign secondary structural elements within the core region
and (c) investigate the reported parallelism within α-synuclein fibrils (Der-
Sarkissian et al. 2003).

3.3 Experimental Procedures
Generation of α-Synuclein Single-Cysteine Mutants
A truncation mutant (115ter) of human α-synuclein, containing residues 1-115,
was generated by adding two stop codons between residues 115 and 116. The
original wild-type human α-synuclein construct (in pRK172) that was used for this
modification was provided by Dr. M. Goedert (Medical Research Council
Laboratory, Cambridge, UK). No native cysteines exist in human α-synuclein.
Single cysteines were introduced by site-directed mutagenesis and verified by
DNA sequencing.

32
Protein Expression and Purification of 115ter α-Synuclein
Proteins were expressed and purified based upon the previously described
protocol for the full-length protein (Der-Sarkissian et al. 2003). The only notable
difference was that the steps after acid precipitation and centrifugation were
modified. At this point, the 115ter proteins were dialyzed against 20 mM MES,
pH 6.0, 1 mM DTT and 1 mM EDTA, then loaded onto a HiTrap SPXL column
(Amersham Biosciences, GE Healthcare) that was equilibrated with the same
buffer. Proteins were then eluted in 0 - 1 M NaCl gradient and fractions were
analyzed by SDS-PAGE. Fractions containing 115ter α-synuclein were
subsequently pooled and 1 mM DTT was added. Protein purity was > 95%
according to Bio-Safe
TM
Coomassie Stain (Bio-Rad). Protein concentration was
determined by using a Micro BCA protein assay kit (Pierce).

Spin Labeling and Fibril Assembly of 115ter α-Synuclein
Recombinant α-synuclein was filtered through an Amicon YM-100 spin filter
(MWCO 1×10
5
, Millipore) to remove any pre-aggregates. Immediately before
spin labeling, DTT was removed by loading the protein solution onto a PD-10
column (Amersham Biosciences, GE Healthcare) equilibrated with buffer
containing 20 mM Hepes, pH 7.4, 100 mM NaCl and 1 mM EDTA, then eluted
using the same buffer. α-Synuclein was labeled with 10-fold molar excess of R1
MTSL spin label [1-Oxyl-2,2,5,5-tetramethyl-D-pyrroline-3-methyl]-
methanethiosulfonate (Toronto Research Chemicals, Toronto, Ontario, Canada)
or with a mixture of diluted R1 spin label and the diamagnetic analog, R1’ [1-

33
Acetyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl]-methanethiosulfonate (Toronto
Research Chemicals) (see Figure 3-1) for 1 hour at room temperature. Excess
label was removed using PD-10 columns with the aforementioned elution buffer.
Spin-labeled proteins were washed twice with elution buffer, concentrated using
Amicon Ultra-4 centrifugal filter units (MWCO 5×10
3
, Millipore), and assembled
into fibrils as described previously (Der-Sarkissian et al. 2003). Spin-labeled
proteins (100 μM or higher) were incubated in the aforementioned elution buffer
at 37
o
C under constant agitation for 3 to 5 days. Fibrils were harvested by
ultracentrifugation and washed twice with the same elution buffer. The quality of
the fibrils was monitored by electron microscopy (EM) of negative-stained
samples, by circular dichroism (CD) spectroscopy, and by thioflavin T
fluorescence spectroscopy as described (Jayasinghe et al. 2004). The truncated
synuclein formed fibrils much faster than did the full-length wild- type (8 hours
versus 48 hours). Fibril dimensions were similar to that of wild-type, although
increased lateral aggregation of the 115ter-based fibrils was noted. Seeding R1-
labeled 115ter with full-length wild-type α-synuclein fibrils was performed as
reported previously (Wood et al. 1999).

Figure 3- 1. Chemical structure of the paramagnetic side chain R1 and its
diamagnetic analogue, R1’.

X-band EPR Spectroscopy and Data Analysis
The derivatized proteins were loaded into quartz capillaries (0.6 mm inner
diameter × 0.84 mm outer diameter, VitroCom, Mt. Lakes, NJ) or TPX capillaries
(for accessibility measurements), and EPR spectra were recorded on X-band
Bruker EMX spectrometers (Bruker Instruments, Billerica, MA). Full spectral
scans were performed using an ER 4119HS resonator, and accessibility
measurements were performed using an ER 4123D dielectric resonator at room
temperature. The spectral scans obtained at room temperature using the ER
4119HS resonator had a scan width of 150 Gauss at an incident microwave
power of 12 mW. All EPR spectra shown were normalized to the same amount
of spins, using double integration, and are presented as normalized. For
distance measurements, EPR spectra were obtained at 233K using a Bruker N
2

temperature controller (ER4131VT), and were recorded at 200 Gauss scan width.

34

35
The O
2
accessibility π(O
2
) of fibrils from 25% R1-labeled 115ter α-synuclein was
determined using the standard power saturation method (Altenbach et al. 1994).
EPR spectra were recorded as a function of microwave power (from 100.58 to
1.59 mW) in the presence of pure O
2
and N
2
at room temperature. The peak-to-
peak amplitude of the central line was plotted as a function of incident microwave
power and fitted to obtain the P
1/2
parameters for O
2
and N
2
(Altenbach et al.
1994). The dimensionless quantity π(O
2
) was obtained from the difference of the
respective P
1/2
values divided by the peak-to-peak line width of the given sample.
To correct for variations in resonator properties, we also applied the
normalization using the diphenyldipicrylhydrazide standard as described in
reference (Altenbach et al. 1994). Sample stability was verified in control
experiments, which demonstrated that EPR spectra and accessibilities did not
change over the time course of one month. Simulation of dipolar broadening was
performed using software generously provided by Drs. Altenbach and Hubbell, as
described previously (Altenbach et al. 2001).

3.4 Results
Exchange Narrowing Reveals Parallel, In-register Structure with Physical
Contact Between Same Residues in Core Region of α-Synuclein Fibrils.
Previous SDSL studies on α-synuclein have shown that same sites from
different molecules come into close proximity (Der-Sarkissian et al. 2003). The
accuracy of this analysis, however, was not sufficient to fully distinguish whether
parallelism occurred between sheets (~ 10 Å apart) or strands (4.7 Å apart), and

36
it was not clear whether the spin-spin interactions arose from the proximity of two
or more spin labels. With sufficient spectral quality, such a distinction can be
made by EPR as the close proximity between same sites in multiple parallel
strands can give rise to characteristic exchange narrowed, single line EPR
spectra (Margittai et al. 2004; Margittai et al. 2006). Since exchange narrowing is
defined by single-line EPR spectra (Moline et al. 1980), the presence of hyperfine
(outer) peaks can easily obscure this spectral line shape. In order to optimize
spectral quality and minimize components from unpolymerized protein or other
background labeling (possibly due to codon mistranslation (Masuda et al. 2006)),
we employed a C-terminal truncation mutant of α-synuclein, containing residues
1 - 115. The highly negatively charged C-terminus of α-synuclein remained
unstructured in the fibril and was inhibitory to fibril formation in vivo and in vitro
(Crowther et al. 1998; Serpell et al. 2000; Der-Sarkissian et al. 2003; Du et al.
2003; Murray et al. 2003; Hoyer et al. 2004; Hoyer et al. 2004; Del Mar et al.
2005; Heise et al. 2005; Kaylor et al. 2005; Li et al. 2005; Liu et al. 2005). In
agreement with these previous studies, we found that the C-terminal truncation
mutant formed fibrils much more readily. According to thioflavin T fluorescence
and far-UV CD spectroscopy, fibrils formed in ~8 hours as compared with 48
hours for the full-length protein (data not shown). Although the truncation mutant
had a greater tendency to aggregate laterally, its fibrils had a highly similar
morphology to that of wild-type α-synuclein, giving rise to fibril diameters of 6 to
12 nm; this is in agreement with previously published results for wild-type α-
synuclein as well as for C-terminal truncation mutants (Crowther et al. 1998;

37
Murray et al. 2003). We therefore generated singly R1-labeled 115ter derivatives
and used EPR spectroscopy to investigate their structures in both soluble and
fibrillar forms.
Spectra of all 115ter derivatives in solution were highly similar to each
other. As illustrated with the representative examples of 25R1, 52R1 and 103R1
(Figure 3-2A, plotted at ×1/10 scale), the spectra have very sharp and narrowly
spaced lines. These spectral features arise from a high degree of mobility (R1
rotational correlation time in the sub-nanosecond time scale), and are in
agreement with the previously noted disordered structure in solution (Weinreb et
al. 1996; Der-Sarkissian et al. 2003; Bertoncini et al. 2005; Dedmon et al. 2005).
As shown in Figure 3-2B-D, fibril formation induced significant spectral
changes. Spectra derived from sites within the N-terminus (Figure 3-2B: R1-
labeled positions 25, 30 and 32) have multiple components of varying mobility,
which is in agreement with the notion that the structure in this region is likely to
be heterogeneous (Der-Sarkissian et al. 2003; Del Mar et al. 2005). While
spectra from the C-terminus (Figure 3-2D: R1-labeled positions 103, 108 and 109)
are also heterogeneous, yet are dominated by sharp lines of high mobility, these
components are still broader than those from the soluble protein (for example,
compare position 103 spectra between 2A and 2D). Overall, the data from the C-
terminal sites are consistent with the previously reported presence of highly
dynamic structure in this region (Der-Sarkissian et al. 2003; Del Mar et al. 2005;
Heise et al. 2005).

Figure 3- 2. EPR spectra of R1-labelee α-synuclein (115ter) in soluble and
fibrillar forms.
(A) EPR spectra of freshly prepared, predominantly monomeric spin-labeled
derivatives harboring R1 at the indicated positions. (B-D) EPR spectra of spin-
labeled derivatives in fibrillar form. Spin labels were introduced at the indicated
positions in the N-terminal region (B), in the core region (C), or at the C-terminal
sites (D). All EPR spectra were obtained at room temperature using a 150
Gauss scan width and were normalized to the same number of spins. Due to the
large difference in amplitude, the spectra for soluble α-synuclein (A) are shown at
reduced size (x1/10).

38

39
Most spectra derived from sites located within the core region (Figure 3-
2C: R1-labeled positions 52, 60, 71, 80 and 90) are almost completely free of
hyperfine lines (the remaining outer peaks amounts to less than 0.3% of the
signal, as estimated by spectral simulations). The single-line EPR spectra
obtained from these sites are reminiscent of those previously recorded from tau
fibrils and clearly indicate the presence of spin exchange narrowing. Similar
results were also obtained when R1-labeled 115ter derivatives were seeded with
full-length wild-type α-synuclein fibrils (data not shown), suggesting that the full-
length fibrils and 115ter α-synuclein adopt similar fibrillar structures. Thus, these
data argue that, α-synuclein fibrils must generate a highly specific structure in
which multiple, equivalent residues from different polypeptide chains come into
direct contact.
To further investigate the effects of spin exchange narrowing, and to
illustrate that such exchange narrowing requires simultaneous contact between
multiple spin labels, we performed a series of spin-dilution experiments. In this
set of experiments, fibrils were assembled from protein labeled at position 52
with various mixtures of R1 and its diamagnetic analogue, R1’ (Figure 3-1). The
rationale for this spin-dilution experiment is that the presence of R1’ will increase
the distance between R1 groups and thereby decrease the effects of spin-spin
interactions, in particular that of exchange narrowing. As shown in Figure 3-3A
(the integrals/absorption spectra are shown in Figure 3-3B), increasing dilution
with R1’ successively promotes the formation of three-line EPR spectra with clear
hyperfine structure and the resulting spectral features that are indicative of strong

40
immobilization. These data clearly show that the single-line EPR spectra
observed for the fully R1-labeled fibrils are caused by spin-spin interactions.
Next, we systematically investigated the effect of spin dilution on the EPR
spectra of 52R1/R1’ fibrils (Figure 3-3A) by recording the normalized spectral
amplitude as a function of spin dilution (Figure 3-3C, black dots connected by
solid line). At lower percentages of R1 ( ≤ 50%) the dipolar spin-spin interaction
increases with increasing amounts of R1 and causes progressive line and
spectral broadening as well as a loss in signal amplitude. This behavior is
typically observed for dipolar broadening (Langen et al. 1998; Gross et al. 1999).
The continued reduction in signal amplitude that one might expect for higher
percentages of R1 in the case of purely dipolar interaction is schematically
illustrated by the dashed line in Fig. 3C. In the present case, however, the EPR
spectra become narrower at higher percentages of R1, lose the hyperfine peaks
and some of the dipolar broadening, and consequently regain in amplitude
(Figure 3-3C). Thus, exchange narrowing becomes more predominant as more
and more spin labels are coming together.

Figure 3- 3. Spin-dilution experiments of α-synuclein (115ter) fibrils
containing varying mixtures of R1 and R1’ labels at position 52.
(A) Overlay of fibril first-derivative spectra from proteins labeled with the following
percentages of R1: 100% (blue), 90% (cyan), 75% (magenta), 40% (red), 25%
(green) and 15% (black). The remainder was labeled with R1’. All first-derivative
EPR spectra were obtained at room temperature using a scan width of 150
Gauss and were normalized to the same number of spins, and are presented
normalized. While the spectral lines are dominated by exchange narrowing at
high R1 percentages, lower percentages indicate dipolar broadening. (B)
Overlay of fibril absorption spectra generated from normalized first-derivative
EPR spectra (from panel A) by integration. (C) Central line amplitudes of
normalized EPR spectra as a function of R1 percentage (black dots connected
by solid line). The dashed line schematically illustrates the continuous decrease
in amplitude that would be observed, if only dipolar coupling were present.

41

42
It has been well established that dipolar broadening interactions can be
described by a Pake pattern-type broadening function, which can also be used to
obtain interspin distances (Pake 1948; Rabenstein et al. 1995; Altenbach et al.
2001). We therefore tested whether such an analysis could also be used to
describe the broadening that occurs at low percentages of R1 (Figure 3-4).
Using the method described by Altenbach et al. (Altenbach et al. 2001), we
deconvoluted the spectra of α-synuclein fibrils that were obtained at 40% R1 with
those obtained at lower R1 percentages (10 or 15%; Figure 3-4A, left column) to
directly obtain a broadening function that is model-independent (Figure 3-4A,
red trace in center column). The broadening functions for all of these derivatives
are similar and indicate a broad range of distances. The defined peaks and
shoulders suggest that the range of distances is made up of subsets of distances.
Fitting to a weighted sum of Pake functions did indeed reveal that these peaks
correspond to distances of <7 Å, ~ 10 Å, ~ 14 Å, and ~ 19 Å for all of the
derivatives (Figure 3-4A, right column). To further test the validity of the fit, the
spectra obtained at low R1 percentages were convoluted with the fitted Pake
functions. Although minor deviations could, in principle, arise from residual
exchange narrowing or orientation effects (Hustedt et al. 1997), the resulting
spectra (Figure 3-4A, green traces, left column) overlay well with those obtained
at 40% R1, which shows that the Pake functions appropriately describe the
dipolar broadening under the present conditions. Since we had previously
observed exchange narrowing in tau fibrils, we next tested whether the same
approach could be applied to spin dilutions for tau fibrils. As shown for tau fibrils

43
labeled at position 308, analogous results were obtained (Figure 3-4B).
Interestingly, the distance peaks for the α-synuclein and tau fibrils correspond
approximately to the distance between two, three, four and five ß-strands
measured by fiber diffraction studies (Serpell et al. 2000; von Bergen et al. 2001;
Berriman et al. 2003).
The data presented in Figure 3-2C and Figures 3-3 and 3-4 support the
idea of a parallel arrangement in which same residues come into close contact.
In order to investigate whether this behavior applied to all residues in the core,
we performed a systematic nitroxide scanning experiment in which each residue
between positions 34 and 101 was replaced by R1, one amino acid at a time.
Fibril formation from these derivatives was verified by EM imaging and their
structural properties were investigated by EPR spectroscopy. As shown in
Figure 3-5, strongly exchange-narrowed EPR spectra were observed for nearly
all sites (Figure 3-5, red spectra). These data show that, in α-synuclein fibrils,
extended regions are arranged in a parallel, in-register manner that requires
multiple molecules to stack on top of each other.

44
Figure 3- 4. Quantative analysis of dipolar spin-spin interaction at low
percentages of R1.
Panel (A) shows the data and analysis from fibrils of α-synuclein (115ter)
derivatives labeled at the indicated positions. EPR spectra of frozen samples
(233K) labeled with 40% R1 (red traces) and 10% R1 (black traces; 15% in the
case of 79R1) are compared in the left column. The red traces in the center
column were obtained by deconvolution of the aforementioned spectra according
to previously published methods (Altenbach et al. 2001). By fitting this
experimentally obtained broadening function to a set of Pake broadening
functions (black traces in center column), it was possible to convert the
underlying dipolar interaction into the distance distributions given in the right
column. The correspondence between the peaks and shoulders in the
broadening functions and distance is illustrated by the arrows shown in the 90R1
derivative. The green spectra (left column) were obtained by applying the Pake
broadening function (center column, black trace) to the black spectra (left
column). The line shape for the green, Pake pattern-broadened spectrum
coincides well with the experimentally observed spectrum for 40% R1 (red traces,
left column), indicating that the set of Pake functions adequately describes the
dipolar broadening. Panel (B) represents an analogous distance analysis using
EPR spectra obtained previously (35) for spin-labeled tau fibrils labeled at
position 308. Compared are data from 10% and 25% R1-labeled proteins. All
EPR spectra are shown at a scan width of 200 Gauss and are normalized to the
same number of spins.
Figure 3-4: Continued

45

46
Despite the strong prevalence of exchange-narrowed spectra, a few sites
exhibited a pronounced hyperfine structure (outer peaks), suggesting that they
do not take up a stacked structure. These spectral features were most notable at
the beginning or end of the scan (34, 99 - 101), as well as in a region at the
center of the scan (62 - 67) (Figure 3-5, gray boxes). To a much lesser extent, a
hyperfine structure is also present in the spectra of other sites (35, 36, 45, 47 -
49, 58 - 59, 85, 94), but the overall line shape for these sites is still dominated by
exchange narrowing (~ 90% as estimated by spectral simulations).
In an effort to test whether exchange-narrowed EPR spectra could be
observed for sites outside the core region, we generated nine additional
derivatives harboring spin labels at various N-terminal sites. None of the fibrils
from any of these derivatives gave evidence of significant exchange narrowing
(Figure 3-6, red spectra). Thus, unlike most regions of the fibril core, the N- or C-
terminal regions do not have the same pronounced stacked, in-register
organization.

Figure 3- 5. EPR spectra of α-synuclein (115ter) fibrils containing single
labels at positions 34 – 101.
Spectra from fibrils labeled with 100% R1 are shown in red. Spectra from
samples labeled with a mixture of 25% R1 and 75% R1’ are shown in green.
Gray boxes highlight sites with significant hyperfine splitting in spectra from
100% R1-label. All EPR spectra were obtained at room temperature using a
scan width of 150 Gauss, and were normalized to the same number of spins.

47

Figure 3- 6. EPR spectra of α-synuclein (115ter) fibrils containing single
labels at selected sites in the N- and C- terminal regions.
Spectra from fibrils labeled with 100% R1 are shown in red. Spectra from
samples labeled with a mixture of 25% R1 and 75% R1’ are shown in green. All
EPR spectra were obtained at room temperature using a scan width of 150
Gauss and normalized to the same number of spins.

Structural Features of α-Synuclein Fibrils from R1 Mobility and
Accessibility.
R1 mobility has been shown to be a sensitive indicator of local structure.
Therefore, we sought to use R1 mobility to obtain additional structural information
for α-synuclein fibrils. Since the EPR spectra from fully-labeled fibrils are
affected by mobility as well as by spin-spin interactions (Hubbell et al. 2000), we
used the spin-dilution approach to diminish the effects of spin-spin interactions in
order to focus on mobility. For each of the derivatives, we grew fibrils from a

48

49
mixture of 25% R1 and 75% R1’, and recorded their EPR spectra (Figure 3-5 and
Figure 3-6, green spectra). Under these conditions, mobility can be expressed
by the semi-quantitative parameter, the inverse central line width ( ΔH
0
-1
)
(McHaourab et al. 1996). As shown in Figure 3-7A, there is a pronounced
difference in ΔH
0
-1
between the more mobile sites within the N- and C-terminal
regions (Figure 3-7A, gray boxes) and the strongly immobilized sites in the core
region of the fibrils. Nearly all sites between residues 36 and 98 have very low
values that are comparable to those observed for the fibril cores of tau and Aß
(Torok et al. 2002; Margittai et al. 2004). Thus, as in those proteins, the fibril
core of α-synuclein appears to be tightly packed. The most notable exception is
a region encompassing residues 62 to 67 (Figure 3-7A, gray box). Mobility
values in this region are much higher, suggesting a less ordered and less tightly
packed local structure. Residues 83 to 87 and residues 46, 58 also display
elevated mobility, but to a much lesser effect. Sites near the beginning (34 - 35)
or the end (99 - 101) of the core region seem to be located in transitional regions,
as their mobility gradually transitions from low mobility in the core to higher
mobility in the N- and C-termini (Figure 3-7A, gray boxes). Overall, these
mobility values correlate well with the existence of exchange narrowing, since
sites that exhibit exchange narrowing in the fully-labeled state are generally of
lowest mobility.

50
Figure 3- 7.Graphic illustration of EPR data as a function of residue number.
(A) Mobilities and O
2
accessibilities of fibrils containing 25% R1. Mobility is
represented by the inverse central line widths ( ΔH
o
-1
) (black dots, left Y-axis), and
O
2
accessibility is given by π (O
2
) (red triangles, right Y-axis). Both parameters
are plotted as a function of labeling position. Data points are connected by solid
lines in the case of consecutive residues; dashed lines are used otherwise. Gray
shading indicates areas with increased mobilities and accessibilities. Green
shading indicates regions in which π (O
2
) exhibits a periodicity of 2 (at least four
consecutive residues). The top panel gives the α-synuclein sequence from
residues 35 to 100 using the aforementioned color code. (B) Schematic
summary of EPR data. The top panel indicates the location of sites that have
significant hyperfine structure (at more than four consecutive sites) in fibrils
labeled with 100% R1. The panels below highlight the location of residues with
elevated mobility or O
2
accessibility as described in A. The bottom panel
highlights the location of regions in which the O
2
accessibility oscillates with a
factor of 2. A periodicity of 2 indicates that protected and more exposed sites
alternate. Such a periodicity would be expected in the case of ß-sheets that are
preferentially more exposed on one side. No obvious periodicity would be
expected in sheets in which both sides are protected. Thus, the lack of
periodicity in parts of the core region does not necessarily indicate the absence
of ß-structure.

Figure 3-7: Continued

51

52
Although α-synuclein fibrils have significant ß-sheet content, the mobility
data do not exhibit regions of strong periodicity. For soluble globular proteins, a
periodicity of two is commonly observed in ß-sheets wherein exposed and buried
residues alternate. Such a structure requires that one side of a sheet is solvent-
exposed while the other is buried. The lack of periodicity in the α-synuclein fibril
core therefore indicates the formation of sheets with packing interactions on both
sides (possibly due to a combination of intrasheet, intersheet and interfilament
interactions). It is possible, however, that a facet of ß-sheets in α-synuclein fibrils
may be more surface-exposed as well as more accessible to O
2
. To test this
idea, we equilibrated the fibrils labeled with 25% R1 with pure O
2
(at atmospheric
pressure, see Experimental Procedures) and determined the oxygen accessibility
(expressed as π (O
2
)). As shown in Figure 3-7A (red triangles, right Y-axis), the
same regions that have elevated mobility (N- and C-termini, as well as residues
62 - 67) also have the greatest accessibilities. Our data illustrated that these
more loosely packed regions are more readily accessible to O
2
. Although
accessibility is generally low at most sites in the core region, we observed small
periodic oscillations with a periodicity of two in several regions. These regions
include residues 35 - 40 (EGVLYV), 51 - 54 (GVAT), 69 - 82
(AVVTGVTAVAQKTV), 83 - 87 (EGAGS), and 95 - 98 (VKKD) (green boxes and
green dashed box in Figure 3-7A; residues facing outward are highlighted in
aforementioned bold underlined letters), indicating the potential of a ß-sheet
structure in these regions (also see Discussion).

53
3.5 Discussion
The goal of the present study was to obtain more detailed structural
information on the core region of α-synuclein fibrils. Toward this end, we
generated 83 spin-labeled derivatives, including a complete nitroxide scan from
positions 30 to 101, and studied the structural features of these derivatives using
EPR spectroscopy.
Mobility and accessibility analyses revealed a tightly packed region
extending from residue 36 to residue 98, with only few localized areas of
elevated mobility and accessibility. Importantly, most sites within this range
exhibited exchange-narrowed EPR spectra that were dominated by single lines
with little or no detectable outer peaks. As illustrated in the spin-dilution
experiments, these spectral lines were present only under conditions in which a
large number of spin labels came into contact with each other. The close
proximity of two labels at a time can give rise to an exchange interaction that
causes a five-line EPR spectrum, and this has been observed for small biradicals
and bilabeled peptides with nitroxides in close proximity (Luckhurst 1966;
Hanson 1996). Single-line, exchange-narrowed EPR spectra, however, are very
rare in spin-labeled proteins, as they require the orbital overlap of several labels.
No single-line EPR spectra have been observed for proteins in which two R1
labels were in close proximity. This includes a pair of spin labels that were within
van-der Waals contact in a crystal of T4 lysozyme (Langen et al. 2000). In this
case, physical contact was made by the flanking methyl groups, resulting in a
nitroxide-nitroxide distance of 8.1 Å. Although the close proximity is reflected by

54
strong dipolar broadening in the EPR spectrum, there is no evidence of any
exchange interaction (neither exchange narrowing nor a five-line EPR spectrum).
Furthermore, studies in which three (Gross et al. 1999) or four (Langen et al.
1998) spin labels came into close contact did not reveal any significant exchange
narrowing. In crystals of spin labels, however, wherein an indefinite number of
nitroxides are stacked on top of each other, such exchange narrowing is
commonly observed (Lajzerowicz-Bonneteau 1976). The distance between the
spin labels in such crystals as well as in α-synuclein must be close enough to
allow averaging of the hyperfine interaction. Thus, the frequency of the
exchange interaction must be much greater than that of hyperfine interaction (>>
10
8
s
-1
). The Pake pattern-based convolution/deconvolution method of Fig. 4
indicates that multiple spin labels are tightly packed together at a distance close
to that of the inter-strand distance derived from fiber diffraction (4.7 to 4.8 Å).
The distance range of continuous wave EPR spectroscopy ( ≤ 20Å) was sufficient
to measure distances between spin labels located on up to five strands apart
from each other. While larger distances could not be detected using the current
methodology, the data are nevertheless consistent with a model wherein an
indefinite number of spin labels are stacked along the fibril axis (Figure 3-8).
In summary, the EPR spectra demonstrate that α-synuclein fibrils must be
arranged in a parallel, in-register structure in which multiple polypeptides stack
on top of each other. Overall, these data are in good agreement with the
emerging view that parallel arrangement is a rather common feature shared by a
number of fibrils; these include Aß, IAPP, a short fragment of ß2-microglobulin,

55
and the yeast prions Sup35p and Ure2p
10-39
, as well as crystals of short peptide
fragments from amyloidogenic proteins (Benzinger et al. 1998; Antzutkin et al.
2000; Torok et al. 2002; Jayasinghe et al. 2004; Chan et al. 2005; Iwata et al.
2006; Shewmaker et al. 2006; Sawaya et al. 2007). Given the fact that ß-strands
within the core region of α-synuclein (and other amyloid) fibrils are arranged
perpendicular to the fibril axis (Figure 3-8), a parallel, in-register structure
requires that each layer of the fibril (every 4.7 to 4.8 Å, Figure 3-8) must contain
a new polypeptide molecule. Such a structure maximizes intermolecular contact
surface, a feature that is likely to aid in the highly specific fibril propagation (Der-
Sarkissian et al. 2003; Margittai et al. 2004).
Another unique feature of fibrils with parallel, in-register structure is that
their stability is strongly affected by the packing interactions between same
residues that stack on top of each other (Margittai et al. 2006). A recent study on
tau fibrils demonstrated that the stacking interactions of charged residues are
strongly destabilizing, while the stacking of ß-branched residues, such as Ile and
Val, is highly stabilizing. In this respect, it is noteworthy that α-synuclein contains
a repeat region found to be critically important for fibril formation (residues 71 to
82) (Giasson et al. 2001; Pawar et al. 2005). This region, which is not present in
the less amyloidogenic ß-synuclein, differs from other repeats in that it is devoid
of charged residues and instead contains a high proportion of hydrophobic ß-
branched residues (see sequence in Figure 3-7A). Thus, the increased
amyloidogenic nature conferred by this region can readily be rationalized by the
extensive interactions that occur between same residues in parallel, in-register
structures.

Figure 3- 8. Schematic illustration of α-synuclein fibril structure
highlighting the parallel, in-registrer arrangement of multiple strands.
The outer cylinder schematically illustrates the overall fibril, and the inner
cylinders represent the individual filaments within the fibril. Strands within one of
those filaments are represented by green arrows. According to fiber diffraction
(Serpell et al. 2000), α-synuclein takes up a cross-ß structure in which ß-strands
run perpendicular to the fiber axis (4.7 ~ 4.8Å apart). The EPR data show that a
spin label introduced at a given site within the core region (schematically
indicated by orange circles) stacks on top of equivalent sites in neighboring
polypeptides. The close contact between same residues at sites throughout the
core region demonstrates a parallel, in-register structure in which multiple
strands run parallel to each other. Importantly, each layer contains a new
molecule. Such a structure maximizes inter-molecular contact surface, a feature
that is likely to aid in the highly specific fibril propagation.

56

57
Although the core region of α-synuclein fibrils is characterized by
pronounced stacking interactions and strong immobilization, there are some
regions that do not experience such a structure. The most pronounced lack of
stacking interactions and highest mobility were observed for the region around
residues 62 to 67 (Figure 3-5, gray box; Figure 3-7A, gray box; Figure 3-7B, top
two panels). These data suggest that this region, which coincides with the
beginning of the NAC region, represents a less tightly packed region that is
probably in a loop or turn conformation. Increased mobility and loss of stacking
interactions were also observed at residues 34, 35 and 99, which are located at
the N- and C-terminal boundaries of the core region (Figure 3-7A, gray boxes;
Figure 3-7B, top two panels).
While mobility information clearly revealed that the aforementioned
regions are of higher mobility, it did not directly reveal the location of the ß-sheet
structure. Such an assignment can readily be accomplished when a given ß-
sheet is exposed on one side and buried on the other side (Hubbell et al. 1998).
The fact that no such periodicity could be observed in α-synuclein fibrils indicates
that significant packing interactions must be present on all sides of the ß-sheets
within the fibril. By performing accessibility measurements using 100%
molecular O
2
, however, we were able to increase sensitivity. The accessibility
data served to identify several regions with a periodicity of two, which is
characteristic of ß-structure (Figure 3-7A, green boxes: 35 - 40, 51 - 54, 69 - 87
and 95 – 98; Figure 3-7B, bottom panel). The most extensive periodicity was
found for residues 69 - 87, which contain the key repeat region that is absent in

58
ß-synuclein. ssNMR has also suggested ß-structure in this region, although there
was indication that its C-terminal end might be in a bend or turn conformation
(Heise et al. 2005). It should be noted that we observed some, albeit a rather
small, increase in overall mobility for residues 83 - 87 (Figure 3-7A, dashed
green box), as well as the presence of some hyperfine structure at residue 85.
These observations would be consistent with the formation of a more loosely
stacked structure in this region. Although additional data are required to pinpoint
the precise end of the ß-structure, we can clearly define which face of the ß-
structure in this region is more exposed to O
2
(69AVVTGVTAVAQKTVEGAGS87,
highlighted in aforementioned bold underlined letters).
It has recently been suggested that the N-terminal portion of the core
region could have different structures depending upon fibril morphology (Heise et
al. 2005). While structural heterogeneity might well exist in this region, it is
important to point out that exchange narrowing was observed throughout the
core region. Thus, the parallel, in-register orientation must be maintained in all
structures.
Collectively, our EPR data, together with information from other structural
techniques, have provided significant structural constraints for α-synuclein fibrils.
With additional inter-residue distance information from ssNMR and continuous
wave as well as pulsed EPR, it should ultimately be possible to obtain reliable
three-dimensional models or structures.

59
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Rabenstein, M. D. and Shin, Y. K. (1995). "Determination of the distance
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Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A.,
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amyloid cross-beta spines reveal varied steric zippers." Nature 447(7143): 453-7.

Serpell, L. C., Berriman, J., Jakes, R., Goedert, M. and Crowther, R. A. (2000).
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beta conformation." Proc Natl Acad Sci U S A 97(9): 4897-902.

65
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Soto, C. (2003). "Unfolding the role of protein misfolding in neurodegenerative
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66
CHAPTER 4:
DISTANCE MEASUREMENT OF α-SYNUCLEIN FIBRIL BY SITE-DIRECTED
SPIN LABELING

4.1 Abstract

Despite intense efforts, the precise structure of α-synuclein fibrils remains
unclear. In Chapter 3, we have found that the core region of α-synuclein fibrils
was tightly packed and extended from residues 36 to 98. The structure within the
core region was packed parallel and in-register, with same residues from
different molecules stacking on top of each other. Only few areas were
significantly less ordered. Oxygen accessibility indicated the possible location of
potential ß-sheet structures.
Here we continued our effort to further define the structure of α-synuclein
fibrils using continuous wave (cw) and pulsed EPR spectroscopy coupled with
site-directed spin-labeling. We have constructed a battery of double-cysteine
mutants covering most of regions of α-synuclein molecule, including the region
having potential ß-structures (residues 70 to 80), in which two residues were
labeled at the same time. We have successfully established and validated the
feasible method for intra-molecular distance determination. Intra-molecular
distances were obtained from cw and pulsed (4-pulse DEER) EPR
measurements and confirmed that some of the regions indeed form ß-structures.
Our current results have provided us some distance constraints that will allow us
eventually generate an accurate three-dimensional model of α-synuclein fibril,
together with the help from molecular modeling in the near future.

67
4.2 Experimental Procedures
Generation of α-Synuclein double-Cysteines Mutants
A truncation mutant (115ter) construct in pRK172 of human α-synuclein,
containing residues 1-115, was used for the modifications. Pairs of cysteines
were introduced by site-directed mutagenesis and verified by DNA sequencing.

Protein Expression and Purification of 115ter α-Synuclein
Proteins were expressed and purified followed the previously described protocol
for the single-cysteine 115ter mutants (Chen et al. 2007).

Spin Labeling and Fibril Assembly of 115ter α-Synuclein
Proteins were spin labeled and allowed to assemble into fibrils using the
previously described protocol for the single-cysteine 115ter mutants (Der-
Sarkissian et al. 2003; Chen et al. 2007).

Processing a-synuclein fibrils samples for DEER measurements
15% or less spin labeled double-cysteine 115ter α-synuclein fibril pellet sample
was resuspended to ~ 50-60ul final volume in buffer containing 20 mM Hepes,
pH 7.4, 100 mM NaCl and 1 mM EDTA, and load to a round quartz capillary (Cat
# CV1518q, 1.5 I.D. x 1.8 O.D, VitroCom Inc., Mt. Lks. NJ 07046). Freeze the
capillary for overnight lyophilization. In next day, centrifuge the capillary and add
30% sucrose on the top of sample and spin again for couple of more times to
bring down all the samples. Usually 10ul of 30% sucrose was enough to let fibrils

68
immerse in it but could vary depends on the specific fibrils sample volume. Flash
frozen the capillary in liquid nitrogen and store at -80
o
C for later DEER signal
measurement.

X-band EPR Spectroscopy and Data Analysis
EPR spectra were recorded on X-band Bruker EMX spectrometers (Bruker
Instruments, Billerica, MA). Full spectral scans were performed using an ER
4119HS resonator at -233K, with scan width of 200 Gauss at an incident
microwave power of 12 mW. All EPR spectra shown were normalized to the
same amount of spins, using double integration, and are presented as
normalized. For distance measurements, EPR spectra were obtained at 233K
using a Bruker N
2
temperature controller (ER4131VT), and were recorded at 200
Gauss scan width. Simulation of dipolar broadening was performed using
software generously provided by Drs. Altenbach and Hubbell, as described
previously (Altenbach et al. 2001).

Initial DEER measurements and Data Analysis (Performed at Univ. of Pittsburgh,
Collaborated with Professor Sunil Saxena)
The DEER experiments were conducted using Bruker EleXsys E580 CW/FT X-
band ESR spectrometer equipped with a Bruker X-band ER 4118X-MS2 split ring
resonator and 4118X-MS3 split ring resonator. Both resonators provided
identical results for the same measurement. The temperature was controlled by
an Oxford ITC605 temperature controller and an ER 4118CF gas flow cryostat.

69
All experiments were performed at the temperature of 80 K. Four-pulse DEER
experiments were obtained with a resonator Q ≤ 100 and an ASE TWTA with an
output power of 1 KW. The pulse sequence for generating the dipolar time
evolution data is shown in Figure 4-1 (Pannier et al. 2000). The time domain was
collected for 64 points with 16 ns time increment (t), with main pulse of 16-32 ns
and ELDOR pulse of 32 ns. The total acquisition time for each sample was
roughly 23 - 24 hours.

The data was initially cut off at 2000 ns and baseline corrected. The background
decay due to intermolecular interaction was subtracted by fitting the last 75% of
the data with first /2
nd
/3
rd
order polynomial function. Occasionally, the data
would be treated with the low pass filter to help get rid of electron-nuclear
interaction ESEEM frequency before it’s cut off at 2000 ns. And the treated data
was further smoothed by Hamming function and zero-filled to 256 or 512 points
in order to enhance signal-to-noise (S/N) ratio. The time domain data was
analyzed using Tikhonov regularization method with a regularization parameter
to determine the distance distribution functions. Fourier Transform of the
processed data provided the frequency spectra.

In-house DEER measurements and Estimation of Intra-Molecular Distance
The in-house DEER experiments were conducted using Bruker EleXsys E580
CW/FT X-band ESR spectrometer equipped with a Bruker 3-mm (MS-3) split ring
resonator, a continuous-flow helium cryostat (CF935, Oxford Instruments), and a
temperature controller (ITC503S, Oxford Instruments). Four-pulse DEER
experiments (see Figure 4-1, (Pannier et al. 2000)) were performed by Dr. B.G.
Hegde in the lab, as described previously (Milov et al. 2003; Jeschke et al. 2004;
Hilger et al. 2005). All experiments were performed at the temperature of 78 K.
The acquisition time used for each sample was roughly 24 hours – 48 hours.

Estimation of intra-molecular distance were fitted by Dr. Ralf Langen using
Tikhonov regularization (Chiang et al. 2005) as implemented in
DEERAnalysis2006 and DEERAnalysis2008 packages (Jeschke et al. 2006) .

Figure 4- 1. Four-pulse DEER sequence.
Schematic description of Pulse patterns for dead-time-free Four-pulse DEER.
The time t between the second and third pulse of the sequence is incremented in
steps of 8 ns. (Adapted from (Pannier et al. 2000) )

70

71
4.3 Results and Discussions
Intra-molecular Distance Measurement on α-Synuclein Double-cysteine
Mutants by X-band Continuous Wave EPR Spectroscopy.
Our Oxygen accessibility data had shed a light on the potential location of
possible ß-sheet structures in the α-synuclein fibrils (Chen et al. 2007). In an
effort to further define its structure, we initially constructed a series of double-
cysteine mutants covering the region having potential ß-structures (residues 70
to 80), in which two residues were labeled at the same time. There are some
caveats in this set of experiments. First of all, in a doubly labeled system, it is
very important to separate intra- and inter-molecular interactions. For such
purpose, sufficiently spin diluted samples would be required, i.e. 15% spin
labeled or even less to avoid spin-spin exchange. A similar problem that also
had been observed in a single labeled system would be some of the spin-labeled
double-cysteine derivatives might not form fibrils; hence it is always necessary to
check fibril formation under EM.
Taken all these into consideration, the intra-molecular distances were
acquired using continuous wave (cw) EPR measurements and the data was
processed using Pake pattern analysis. Our results confirmed that some of the
regions indeed form ß-structures, as shown in Figure 4-2. This set of study
served two purposes, first of all it validated my previous oxygen accessibility data,
and secondly it established the foundation for us to subsequently establish and
test the feasibility of pulsed EPR distance measurement on the α-synuclein fibrils.

72
73/75
Mutants Expected distance
Observed
(main distance)

73/75 7 Å 9 Å
73/76 17 Å 17.5 Å
73/77 14 Å 14 Å
73/76

73/77

Figure 4- 2. Distance measurements in α-synuclein (115ter) fibrils from
continuous wave EPR spectroscopy.
Left panel: Spin dilution (87.5% R1’ labeling and mixed with either 12.5 % R1 or
12.5% of 40% R1 labeling) was used to reduce spin-spin interactions in the
samples. All cw EPR spectra were obtained at room temperature using a 200
Gauss scan width and were normalized to the same number of spins. In all
cases, 87.5% of the double mutants were labeled with non-paramagnetic label.
The remaining 12.5% were either fully labeled with the paramagnetic R1 label
(red traces) or labeled with a mixture of paramagnetic and non-paramagnetic
label at a 2:3 ratio (black trace). Distances were simulated based on Pake
pattern analysis. Right panel: Comparison of expected and observed distances.
Expected distances are based on standard ß-strand geometry and the nitroxide
backbone distances as described (Langen et al. 2000), in which 7 Å is
considered a typical distance between the R1 nitroxide and the backbone.
Distance distribution peaks are given for the experimentally determined distances.

73
Method Establishment for Processing α-Synuclein Fibrils Samples for
DEER Measurements
This sophisticated pulsed EPR technology is so new that, to date, little
information concerning pulsed EPR methods has been published, especially for
amyloid fibrils. To my knowledge, our group was the first to utilize this
technology for the purpose of measuring distances on amyloid fibrils. In this
regard, we needed to establish methods to measure the longer distances on
fibrils by using pulsed EPR.
First of all, to measure intra-molecular distances, we need double-cysteine
mutants. The challenge is which pairs of residues we choose and start with.
How do we prepare the samples for pulsed EPR measurements? How do we
optimize the data acquisition condition and how do we interpret the data? All
those are concerns need to consider during the process of method establishment.
In order to work out the conditions, I decided to test the spin-labeled fibrils
containing two spin labels at a time. I initially focused on a region that I found to
be in a ß-sheet conformation based on my oxygen accessibility data and cw EPR
measurements.
Having tried different spin label conditions and different processing
methods, we finally have been successful in establishing methods of sample
labeling and sample preparations. It is important to note that, after samples were
properly processed as described in the method section, we would measure the
samples again via cw EPR in room temperature to make sure that such
complicated manipulation would not disrupt the samples and the present spin-

74
spin interaction is still intact. Our cw EPR data (data not shown here) did confirm
that our sample preparation did not introduce any disruptive or artificial effect to
the processed samples.

Working Scheme to Design α-Synuclein Double-cysteine Mutants
As mentioned earlier, we initially focused on building our α-synuclein
double-cysteine mutants at NAC region in order to test the validity of our method.
As it turned out to be working, we attempted to build more double-cysteine
mutants. Under the principle of obtaining accurate and sufficient distance
constraints and at the same time be efficient, we utilized the following working
scheme to design and construct our double-cysteine mutants, as shown in Figure
4-3. Basically, we need cover the entire core region. We would use the same
reference sites as internal controls to obtain distances from different test sites at
different regions ( β-strand/turn). For each site, 3 or more distances are required
to confirm and validate its position. Also we try to minimize the tests through
overlapping the adjacent test regions. Finally we would introduce some external
controls, such as measuring the distance outside of the core region if possible.
Currently available double cysteine-mutants have been summarized in Table 4-1.

75

N-term
C-term

Figure 4- 3. Schematic illustration of the experimental design to generate α-
synuclein double-cysteine mutants for potential distance measurements.

76
Tabel 4- 1. Summary of available α-synuclein double-cysteine mutants

Reference Site Test Site Reference Site Test Site
19 24 69 49
36 44

59

69

60

87

73
37 48

77
38 53

79

84 73 53

90

69
39 84

75
40 97

76
41 87

77
46 59 77 53
49 69

69
52 55

73
53 38

84

61

87

72

89

73 80 93

74

94

77 83 89

87

90

94

91
97 84 38
54 73

39
55 52

77
56 100 87 36
57 97

41
60 69

53

87

60
61 53 89 77
69

83
94

98
94 53
97 40

53
98 87
89
95

77
Inter-molecular Distance Measurement on α-Synuclein Single-cysteine
Mutants by Pulsed EPR Spectroscopy.
Our data have demonstrated that α-synuclein molecules are arranged in a
parallel fashion in fibrils, with same residues from different strands stacked on
top of each other with very close contact (Chen et al. 2007). However, the exact
inter-molecular distance for α-synuclein molecules in fibrils is still unclear. X-ray
fiber diffraction indicate ß-strands are separated 4.7-4.8 Å apart (Serpell et al.
2000). We know that the distance constraint of spin exchange is < 8 Å. Such
distance constraint cannot be estimated by lineshape analysis of continuous
wave (cw) EPR spectra, because it is below the distance range of dipolar
interaction (8-20 Å) that can be reliably determined by lineshape analysis.
Therefore to obtain inter-molecular distance on single-cysteine mutants, pulsed
EPR can be applied; however the method has not been developed yet, so far no
such distance measurement has been performed on amyloid fibrils. In theory,
pulsed EPR detects distance of 15 - 80 Å through modulation of dipolar
interaction, and multiple inter-spin distances can be obtained simultaneously.
To measure the inter-strand distance in α-synuclein fibrils derived from
single-cysteine mutants, we carried out our initial experimental DEER (double
electron-electron resonance) experiments on pulsed EPR, in collaboration with
Dr. Saxena at University of Pittsburgh. Initially, 100% spin labeled single-
cysteine mutants were tried but DEER failed to obtain any distance. Because the
presence of spin-exchange causes molecules stacked on top of each other very
closely, the inter-molecular interactions are so dominant that lead to quick

78
baseline decay. And such baseline decay needs be subtracted during the
distance calculation process. Hence, to lower the effect of spin-exchange and
avoid strong baseline decay, 25% spin labeled samples were utilized in the
DEER experiments, including the fibrils derived from representative sites 25 (at
N-terminal), 52 (at core region), 63 (at loop/turn), 79 (at core region) and 109 (at
C-terminus). There is one caveat in the DEER experiments, that ESEEM
(electron spin-echo envelope modulation) effect due to the hyperfine coupling,
the electron-nuclear dipolar (END) interaction, could appear and interfere the
data analysis. Such effect could be eliminated by optimizing the experiment
conditions and it should be eliminated as a basis of the echo modulation. To
summarize, we found that the inter-strand distance in the core region of α-
synuclein fibrils is ~5 Å, which was in excellent agreement with X-ray fiber
diffraction data. We would expect inter-molecular distance at C-terminus to be
larger than 5Å since C-terminus is unstructured; one at N-terminal might be
varied depending on the residue being tested, since N-terminal is a less ordered
heterogeneous region and some region in N-terminal could refold back into other
structure. The inter-molecular distance in loop/turn might be around 5Å since it is
still part of the core. One thing to note that, in order to obtain reliable distance
information, the experimental condition needs to be optimized; however such
optimization was not sufficient in the aforementioned pulsed EPR measurements
and therefore the data acquired from such experiments was not conclusive at this
point.

79
Intra-molecular Distance Measurement on α-Synuclein Double-cysteine
Mutants by Pulsed EPR Spectroscopy.
To establish and test the feasibility of the pulsed EPR DEER approach for
intra-molecular distance determination, our strategy was to re-test those double-
cysteine mutants we had tested using continuous wave (cw) EPR method. My
preliminary data from both cw EPR and pulsed EPR measurements (data not
shown here) have shown consistency with oxygen accessibility results;
furthermore, my data demonstrated pulsed EPR measurement is indeed a
feasible method. Moreover, my strategy has also or will benefit the work
undertaken by some of my colleagues.
To summarize this part of the study, we have established the cw and
pulsed EPR methods to obtain reliable intra-molecular distances in α-synuclein
fibrils. Using accurate and sufficient distance constraints, it should ultimately be
possible to obtain reliable three-dimensional models or structures of α-synuclein
fibrils. Collectively, this information will assist us in deciphering oligomeric
species and benefit future therapeutics development.

80
4.4 Chapter 4 References
Altenbach, C., Oh, K. J., Trabanino, R. J., Hideg, K. and Hubbell, W. L. (2001).
"Estimation of inter-residue distances in spin labeled proteins at physiological
temperatures: experimental strategies and practical limitations." Biochemistry
40(51): 15471-82.

Chen, M., Margittai, M., Chen, J. and Langen, R. (2007). "Investigation of alpha-
synuclein fibril structure by site-directed spin labeling." J Biol Chem 282(34):
24970-9.

Chiang, Y. W., Borbat, P. P. and Freed, J. H. (2005). "The determination of pair
distance distributions by pulsed ESR using Tikhonov regularization." J Magn
Reson 172(2): 279-95.

Der-Sarkissian, A., Jao, C. C., Chen, J. and Langen, R. (2003). "Structural
organization of alpha-synuclein fibrils studied by site-directed spin labeling." J
Biol Chem 278(39): 37530-5.

Hilger, D., Jung, H., Padan, E., Wegener, C., Vogel, K. P., Steinhoff, H. J. and
Jeschke, G. (2005). "Assessing oligomerization of membrane proteins by four-
pulse DEER: pH-dependent dimerization of NhaA Na+/H+ antiporter of E. coli."
Biophys J 89(2): 1328-38.

Jeschke, G., Wegener, C., Nietschke, M., Jung, H. and Steinhoff, H. J. (2004).
"Interresidual distance determination by four-pulse double electron-electron
resonance in an integral membrane protein: the Na+/proline transporter PutP of
Escherichia coli." Biophys J 86(4): 2551-7.

Jeschke, G. C., V.; Ionita, P.; Godt, A.; Zimmermann, H.; Banham, J.; Timmel, C.
and R.; Hilger, D. J., H (2006). "DeerAnalysis2006 - a comprehensive software
package for analyzing pulsed ELDOR data." Applied Magnetic Resonance 30(3-
4 ): 473-498.

Langen, R., Oh, K. J., Cascio, D. and Hubbell, W. L. (2000). "Crystal structures of
spin labeled T4 lysozyme mutants: implications for the interpretation of EPR
spectra in terms of structure." Biochemistry 39(29): 8396-405.

Milov, A. D., Tsvetkov, Y. D., Formaggio, F., Oancea, S., Toniolo, C. and Raap, J.
(2003). "Aggregation of spin labeled trichogin GA IV dimers: distance distribution
between spin labels in frozen solutions by PELDOR data." J. Phys. Chem. B 107:
13179-13727.

81
Pannier, M., Veit, S., Godt, A., Jeschke, G. and Spiess, H. W. (2000). "Dead-time
free measurement of dipole-dipole interactions between electron spins." J Magn
Reson 142(2): 331-40.

Serpell, L. C., Berriman, J., Jakes, R., Goedert, M. and Crowther, R. A. (2000).
"Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-
beta conformation." Proc Natl Acad Sci U S A 97(9): 4897-902.

82
CHAPTER 5:
INVESTIGATION OF ß-2-MICROGLOBULIN FIBRIL STRUCTURE BY SITE-
DIRECTED SPIN LABELING AND ELECTRON PARAMAGNETIC
RESONANCE

5.1 Abstract

ß-2-microglobulin (ß2m) is a small monomeric ß-sandwich protein that
plays a critical role in cellular immunology. However, it could misfold and form
amyloid fibrils that are involved in some diseases such as dialysis-related
amyloidosis (DRA). Here we applied electron paramagnetic resonance
spectroscopy together with site-directed spin labeling, to obtain the secondary
structural features of ß2m in solution state, in long-straight (LS) fibrillar and
worm-like (WL) fibrillar forms. In this study, we found that major conformational
changes took place after monomers were converted to fibrils. The structural
features we obtained using EPR were in excellent agreement with what observed
in crystal forms of monomers. Furthermore, our study demonstrated that by
having different morphologies, the LS and WL fibrils do own distinct structural
features. In LS fibrils, single-line, exchange-narrowed spectra were observed in
most of the sites located within the fibrils core region, indicating that the core
region of the LS fibrils must take up a parallel, in-register structure in which same
residues from different molecules are stacked on top of each other. In contrast,
the N- and C-terminus of the protein were much less ordered. However, in WL
fibrils, clear single-line, exchange-narrowed spectra were not presented in any of
the sites we studied, suggesting that WL fibrils do not share a similar parallel, in-
register structure with LS fibrils.

83
5.2 Introduction
ß-2-microglobulin (ß2m) is the non-covalently bound light chain of the
class I human leukocyte antigen (HLA class I) and it has a central role in cellular
immunology that it make sure MHC-1 (the class I major histocompatibility
complex) folds and assemblies correctly. ß2m is degraded and excreted in the
kidney. However, if kidney is going through failure, for example in the case of
dialysis-related amyloidosis (DRA), ß2m can not be metabolized correctly and
efficiently, that leads to free ß2m accumulation (up to 60 folds of normal level)
and cause ß2m self-association into amyloid fibrils, and eventually deposited in
the joints of long-term renal dialysis patients (Radford et al. 2005).
ß2m is a small monomeric protein, contains 99 amino acids including 2
native cysteines, and has a molecular weight of around 12 KD. ß2m holds a
seven ß-strands ß-sandwich structure. Out of those 7 strands, strands A, B, D, E
and strands C, F, G form two ß-sheets respectively. The protein is stabilized by
Cysteine 25 from B strand and Cysteine 80 from F strand.
Up to date, there are more than 80 available crystal structures of human
ß2m bound to heavy chain of class I HLA complex (HLAß2m). In contrast, for
monomeric ß2m (M
H
ß2m), there are one solution structure of M
H
ß2m made
available through NMR study under condition of pH 6.6, 30
o
C (PDB code:
1JNJ;(Verdone et al. 2002)); one crystal structure of M
H
ß2m under condition of
pH 5.7, 16
o
C (PDB code: 1LDS; (Trinh et al. 2002)) combined with NMR method,
as well as another crystal structure derived at pH 7.0 that shared the same
structure as the one formed under pH 5.7 (Iwata et al. 2007). The structural

84
difference between the crystal structures of HLAß2m and M
H
ß2m lies in the
residues in D-strand (50-56) and the succeeding loop (Trinh et al. 2002). In
HLAß2m, residues 50-56 form 2 short 2-aa ß-strands that are separated by a 2-
aa ß-bulge; while in M
H
ß2m, 51-56 form a continuous 6-aa ß-strand that
stabilized by E strand, forming a regular anti-parallel ß-sheet. And in M
H
ß2m,
residues 50-57 are highly ordered in the crystal structure but dynamic in the
solution structure.
In terms of fibrils formation, it was rarely known how the precursors were
populated from the native proteins at the physiological condition. However, in
vitro, the precursor formed at pH 2.5 is more highly unfolded. It is noted that
partial unfolding is a prerequisite for the native proteins to assemble into fibrils. It
was speculated that D strand (aa 50-56) in the crystal structure could work as a
template for amyloid formation because of its long straight conformation. Under
acidic condition, different morphologies types of fibrils are generated (see Figure
5-1), including long-straight (LS) fibrils that have several protofilaments twisted in
a left-handed helical way; flexible and nodular worm-like (WL) fibrils; and the
much shorter rod-like (RL) fibrils formed under moderate ionic-strength buffer but
no salt (Radford et al. 2005).

Figure 5- 1. Morphological types of ß2m fibrils formed in vitro.
(Adapted from (Radford et al. 2005)). A: long-straight (LS) fibrils, B: worm-like
(WL) fibrils, C: rod-like (RL) fibrils.

Different methodologies have been applied to decipher the secondary
structures of the ß2m fibrils, including X-ray fiber diffraction (Smith et al. 2003) ,
AFM (Kad et al. 2003; Gosal et al. 2006), H/D exchange (Hoshino et al. 2002;
Yamaguchi et al. 2004), solution and solid state NMR (Iwata et al. 2006), pepsin
limited proteolysis and ESI-MS (Myers et al. 2006), proline scanning
mutagenesis (Chiba et al. 2003) and trypophan mutagenesis (Kihara et al. 2006),
and etc. The limited proteolysis study on LS fibrils formed under pH 2.5 with low
salt illustrated that aa 1-9 were highly solvent exposed and the rest of the
unfolded monomer aa 10-99 became tightly folded. The H/D exchange study
showed that aa 21-82 formed stable H-bonds in fibrils grown under pH 1.5 and
2.5. NMR study coupled with urea titration found that aa 25-80 was the most
stable region, especially with E strand of aa 60-71 resistant to denaturation. To
summarize, these studies showed that the N- and C-terminus of the fibrils were

85

86
more flexible and the core region was located in the middle stretch, however, the
ends of the cores varied from each other depending on different approaches.
The most recent ssNMR study of 22-residue peptide (K3 peptide) S20-K41
showed that K3 peptide formed fibrils similar to full-length LS fibrils, and the each
K3 molecule was stacked in a parallel and staggered fashion. Nevertheless, the
site-specific structural information of the full length fibrils is still unavailable,
neither is the structural difference between the LS and WL fibrils. In this study,
we utilized site-directed spin labeling and EPR to study the structures of ß2m,
particularly focused on the full length LS and WL fibrils.

5.3 Experimental Procedures
Generation of ß-2-microglobulin Single-Cysteine Mutants, and Protein Expression
and Purification of ß-2- microglobulin
ß-2-microglobulin single-cysteine mutants were generated using site-directed
mutagenesis using the pINK plasmid containing human ß-2- microglobulin gene
in pET23a by Dr. David Smith, and subsequently proteins were expressed and
purified by Drs. David Smith and Carol Ladner at University of Leeds in U.K in a
similar way to the protocol described previously (McParland et al. 2000).

Spin Labeling of ß-2- microglobulin
Recombinant ß-2- microglobulin was dissolved in 25 mM Tris pH 8, 5 mM DTT, 1
mM EDTA for 20 minutes at room termperature. DTT was removed using a Nap
10 column (Amersham Biosciences, GE Healthcare) and proteins were
immediately labeled with 10-fold molar excess of R1 MTSL spin label [1-Oxyl-
2,2,5,5-tetramethyl-D-pyrroline-3-methyl]-methanethiosulfonate (Toronto
Research Chemicals, Toronto, Ontario, Canada) (see Figure 5-2) for 2 hours at
room temperature in the presence of 2.4 Molar Guanidinium HCl. Excess label
was removed afterwards using PD-10 Columns (Amersham Biosciences, GE
Healthcare). Subsequently spin-labeled proteins were purified on a Resource Q
column (Amersham Biosciences, GE Healthcare) and buffer exchanged to Milli-Q
water using Centricon concentrator (MWCO 3 KD) and stored at -20
o
C. MALDI-
TOF/ESI-MS was used to confirm >95% labeling efficiency, as well to confirm
intactness of the native disulfide bond. This was done by Drs. David Smith and
Carol Ladner.

Figure 5- 2. Chemical structure of the paramagnetic side chain R1.
(Adapted from (Chen et al. 2007) ).

87

88
Fibrils Assembly of ß-2- microglobulin
Spin-labeled proteins (0.5mg/ml) were incubated in 25 mM Sodium Phosphate
pH 2.5 with 5% w/w Wild-type fibrils seeds at 37
o
C under constant agitation of
200 rpm on an orbital shaker for 3 to 12 days to allow them to assemble into
long-straight (LS) fibrils. In the presence of 0.4M Salt, spin-labeled proteins were
allowed to grow into worm-like (WL) fibrils. And appropriate ratio of wild-type
proteins was added into spin-labeled proteins to allow them grown into spin-
diluted fibrils, such as 90% or lower percentages labeled fibrils. Fibrils were
harvested by centrifugation at desktop at the maximum speed for 1 hour at 4
o
C
and washed twice with the same incubation buffer. Wild-type fibrils seeds were
prepared from de novo fibrils formed in the same condition as described above
using wild-type proteins (Kad et al. 2001).

Negative-stained Electron Microscopy (EM)
The quality of the fibrils was monitored by electron microscopy (EM) of negative-
stained samples. Fibrils aliquots were diluted 3-5 fold in 3 mM HCl and loaded to
the glow-discharged copper grids coated with colloidion. The grids were blotted
using paper and 4% uranyl acetate was applied afterwards. Images were
captured on a Philips CM10 electron microscope operating at 80 keV. This was
done by Drs. David Smith and Carol Ladner.

89
X-band EPR Spectroscopy and Data Analysis
The derivatized ß2-microglobulin fibrils were loaded into quartz capillaries
(0.6 mm inner diameter × 0.84 mm outer diameter, VitroCom, Mt. Lakes, NJ) or
TPX capillaries (for accessibility measurements), and EPR spectra were acquired
on X-band Bruker EMX spectrometers (Bruker Instruments, Billerica, MA). Full
spectral scans were obtained using an ER 4119HS resonator, and accessibility
measurements were obtained using an ER 4123D dielectric resonator at room
temperature. The spectral scans performed at room temperature using the ER
4119HS resonator were swept for 150 Gauss scan width at an incident
microwave power of 12 mW. All EPR spectra shown were normalized to the
same amount of spins, using double integration, and are presented as
normalized. The O
2
accessibility Π (O
2
) of fibrils from 25% R1-labeled ß2-
microglobulin was calculated using the standard power saturation method
(Altenbach et al. 1994). EPR spectra were measured as a function of microwave
power (from 100.58 to 1.59 mW) in the presence of air and N
2
at room
temperature. The peak-to-peak amplitude of the central line was plotted against
the incident microwave power and fitted to obtain the P
1/2
parameters for O
2
and
N
2
(Altenbach et al. 1994). The dimensionless quantity Π (O
2
) was calculated
from the difference of the respective P
1/2
values divided by the peak-to-peak line
width of the given sample. To correct for variations in resonator properties, we
also applied the normalization using the diphenyldipicrylhydrazide standard as
described in reference (Altenbach et al. 1994).

90
5.4 Results and Discussions
Structural features in solution were consistent with what observed in the
crystal from of M
H
ß2m.
Total of 19 single-cysteine ß2m mutants covering all the regions of the
protein were generated for this study. All ß2m R1-labeled derivatives in original
buffer (Milli-Q water) were initially studied; the spectra (data not shown here)
gave out relatively sharp lines, which were expected for the relatively smaller size
protein (i.e. fast tumbling on the EPR time scale). Therefore we recorded the
spectra again in 30% sucrose to increase the viscosity and reduce the tumbling
to obtain more accurate structural information. Under the condition of pH 7,
25mM NaP with 30% sucrose, the spectra for each site, as shown in Figure 5-3,
become more informative and are in excellent agreement with their location in
the crystal structure of monomeric M
H
ß2m. This was further reflected in the
linewidth measurements, which were summarized in Table 5-1. In the absence
of spin-spin interactions, as should be the case for the soluble protein, these
values are fairly good indicators of local structure. This has been described
predominantly for helical proteins in a number of systems (Margittai et al. 2001;
Isas et al. 2002). Overall, the values obtained for ß2m here were consistent with
what to be expected based upon the structure.

R3 S61

S11 L65

K19
E69

F22 T73

S28 E77

S33 R81

D38 V85

E50 K91

D53 D96

S55

Figure 5- 3. Spectra of 100% R1-labeled ß2m monomers in pH 7, 25 mM NaP
and 30% sucrose.
Individual spectrum was either plotted at x1/2 scale, x2 scale or original scale.
All EPR spectra were obtained at room temperature using a 150 Gauss scan
width and were normalized to the same number of spins.

91

92
Table 5- 1. Comparison of ß2m structural features in M
H
ß2m crystal form,
solution and LS fibrils.

Res# M
H
ß2m (pH
5.7) (Trinh et
al. 2002)
Monomer EPR Spectra
Mobility (pH 7, 25 mM
NaP, 30% Sucrose)
LS Fibrils EPR Spectra
Line shape (pH 2.5, no
salt)
R3 N-terminus
Loop
0.303259 Mobile, hyperfine structure
S11 A strand 0.303860 Mobile, hyperfine structure
K19 A-B Loop 0.413536 Single-line, exchange-
narrowed
F22 B strand 0.278503 Single-line, exchange-
narrowed
S28 B strand 0.223716 Single-line, exchange-
narrowed (less)
S33 B-C loop 0.359123 Single-line, exchange-
narrowed
D38 C strand 0.310312 Single-line, exchange-
narrowed
E50 D-C’ loop 0.257484 Single-line, exchange-
narrowed
D53 D strand (loop
in HLAß2m)
0.487382 Mobile, hyperfine structure
S55 D strand 0.454889 Single-line, exchange-
narrowed
S61 D-E loop 0.278503 Single-line, exchange-
narrowed
L65 E strand 0.332748 Single-line, exchange-
narrowed
E69 E strand 0.413536 Single-line, exchange-
narrowed
T73 E-F loop 0.426875 Single-line, exchange-
narrowed
E77 E-F loop 0.324921 Single-line, exchange-
narrowed (less)
R81 F strand 0.368829 Single-line, exchange-
narrowed
V85 F-G loop 0.290355 Single-line, exchange-
narrowed
K91 G strand 0.213229 Single-line, exchange-
narrowed
D96 C-terminus
loop
0.426459 Mobile, hyperfine structure

93
Exchange Narrowing Reveals Parallel, In-register Structure with Physical
Contact between Same Residues in Core Region of ß2m Long Straight (LS)
Fibrils.
Next, we performed EPR measurements on the Long-straignt (LS) ß2m
fibrils, generated in condition of pH 2.5, no salt. Those spectra were similar to
what we have observed for many other amyloid fibrils (Der-Sarkissian et al. 2003;
Jayasinghe et al. 2004; Margittai et al. 2004; Chen et al. 2007). And it was
evident that major conformational changes must have taken place as illustrated
by the fact that the spectral shape and amplitude for all sites in solution and in
fibrils was very different (also see Table 5-1).
The region contained aa 19 - 91 had very strong spin-spin interactions,
especially positions 19, 22, 33, 38, 50, 55, 61, 69, 73, 81, 85 gave out single line,
exchange narrowed spectra (see Figure 5-4). This single line requires that a
large number of spin labels are physically in contact at the same time (Margittai
et al. 2004; Chen et al. 2007; Margittai et al. 2008). For positions 28, 65, 77 and
91, their spectra were dominated by exchange narrowing though some hyperfine
structures were still present. We concluded that this LS fibril form contains a
parallel, in register core since this trend was observed at many sites in the core
region. However, there was an exception residue 53, whose spectrum exhibited
a pronounced hyperfine structure, suggesting they did not take up a stacked
structure.
The spectra for positions 3, 11 and 96 located at N- and C- terminus were
very different from those having the exchange-narrowed spectra. Although some

94
residual and much weaker spin-spin interaction can be detected, there is little or
no evidence of spin exchange narrowing suggesting that no specific stacking
interaction between same residues occur at the N- and C- terminus. The much
sharper lines obtained at those sites are also in agreement with this notion as
they indicate higher mobility there. Overall, the data agreed nicely with the H/D
and proteolysis data (Myers et al. 2006).
It is also important to mention that, the ß2m Long-straight (LS) fibrils
spectra (Figure 5-5 left panel) were clearly distinct from what obtained from
random precipitates aggregated from the same fibrils using EtOH (Figure 5-5
right panel), as illustrated in the representative sites of D38 and T73.
Further, to explore the nature of the single-line spectra, we performed a
set of spin-dilution experiments at the representative position 61 in LS fibrillar
forms, by mixing R1 labeled fibrils with appropriate portion of unlabeled wild-type
fibrils. Additionally, control experiments had demonstrated that there were no
exchange took place between already formed R1-labeled fibrils and unlabeled
wild-type fibrils (data not shown here). The rationale of the spin-dilution
experiments was that the presence of unlabeled wild-type molecules will increase
the distance between R1 groups and therefore dilute the effects of spin-spin
interactions, particularly exchange-narrowing. As shown in Figure 5-6, with
increasing dilutions, the three-line spectra with clear hyperfine structure (Figure
5-6A) were restored, as well as the amplitude of the central line of the spectra
(Figure 5-6B) went down and further up again. If only dipolar coupling were
present, one would only observe continuous reductions in amplitude.

95

R3 S61

S11 L65

K19 E69

F22 T73

S28 E77

S33 R81

D38 V85

E50 K91

D53 D96

S55

Figure 5- 4. Spectra of 100% R1-labeled ß2m LS fibrils (in pH 2.5, no salt).
All EPR spectra were obtained at room temperature using a 150 Gauss scan
width and were normalized to the same number of spins.

As both α-synuclein and ß2m could form fibrils that contain a parallel, in-
register stacking core (Chen et al. 2007), it is interesting to notice that, the
linewidth of spectra from ß2m LS (long straight) fibrils are comparable to that of
α-synuclein fibrils, as shown in Table 5-2 and Figure 5-7 below.

LS Fibrils EtOH Precipitates

Figure 5- 5. Comparison of spectra of 100% R1-labeled ß2m LS fibrils (in pH
2.5, no salt) versus random precipitates.
Left panel: EPR spectra overlay of ß2m LS fibrils harboring R1 at position 38
(black line) and position 73 (red line). Right panel: EPR spectra overlay of
random precipitates derived from aforementioned ß2m LS fibrils harboring R1 at
position 38 (black line) and position 73 (red line) using EtOH. All EPR spectra
were obtained at room temperature using a 150 Gauss scan width and were
normalized to the same number of spins.

96

0
0. 005
0. 01
0. 015
0. 02
0. 025
0. 03
0. 035
0. 04
0. 045
0. 05
0 20406080
A B

1
% of R1 l abel
Am pl i t ude of cent r al l i ne

00

Figure 5- 6. Spin dilution of R1-labeled ß2m at position 61 in LS fibrillar
form.
(A) Overlay of spin-diluted EPR spectra with 100% (black line), 75% (red line),
50% (green line), 25% (pink line), 10% (blue line) spin-labeled derivatives
harboring R1 at position 61. (B) Amplitudes of central lines (Y-axis) plotted
against the percentage of R1 label (X-axis). All EPR spectra were obtained at
room temperature using a 150 Gauss scan width and were normalized to the
same number of spins.

Table 5- 2. Comparison of EPR spectra central line width (C.L.W.) between
α-synuclein and ß2m.

α-synuclein C.L.W. (in Gauss) ß2m C.L.W. (in Gauss)
52R1 10.62531 22R1 10.55203
60R1 14.87543 69R1 15.38837
71R1 15.82804 73R1 10.3845
80R1 12.89692 81R1 16.70738
90R1 12.75037 85R1 13.40987

97
α-synuclein ß2m

Figure 5- 7. Comparison of EPR Spectra derived from 100% R1-labeled α-
synuclein and ß2m LS fibrils.
Left panel: EPR spectra overlay of α-synuclein fibrils harboring R1 at position 52
(black line), 60 (red line), 71 (green line), 80 (pink line), 90 (blue line). Right
panel: EPR spectra overlay of ß2m LS fibrils (in pH 2.5, no salt) harboring R1 at
position 22 (black line), 69 (red line), 73 (green line), 81 (pink line), 85 (blue line).
All EPR spectra were obtained at room temperature using a 150 Gauss scan
width and were normalized to the same number of spins.

Structural Features of ß2m Long-straight (LS) fibrils from R1 Mobility and
oxygen accessibility.
Mobility and spin-spin interactions are two factors affecting the lineshapes
of EPR spectra (Hubbell et al. 2000). In order to study the effect of mobility, we
performed spin-dilution experiments to eliminate the effect of spin-spin
interactions. Fully R1-labeled Long-straight (LS) fibrils were mixed with
appropriate ratio of unlabeled wild type fibrils to achieve the final 25% R1-label in
the fibrils and such spectra were measured as shown in Figure 5-8. The inverse
central linewidth ( ΔH
0
-1
) was applied to represent the mobility information

98

99
(McHaourab et al. 1996). As illustrated in Figure 5-9, most of the sites in the fibril
core region have relatively low mobility comparing to the sites at N-terminus. It is
noticed that the mobility obtained from the ß2m LS fibrils is relatively higher than
what had been observed in the fibril cores of tau, Aß and α-synuclein (Torok et al.
2002; Margittai et al. 2004; Chen et al. 2007), suggesting the ß2m LS fibrils are
not packed as tightly as those fibrils. The exceptions are positions 53 and 81,
where having higher mobility values, indicating less ordered local structures.
Overall, the mobility information agrees well with the occurrence of exchange
narrowing.
Oxygen accessibility measurement is another important piece of tool to
obtain further structural information for fibrils. Here we equilibrated the LS fibrils
with oxygen at atmospheric pressure and determined the oxygen accessibility π
(O
2
). Amyloid fibrils tend to have high ß-sheet contents, therefore, we could
expect to observe one side of a sheet is more solvent-exposed and the other
side is more buried. And the more solvent-exposed side will be more readily
accessible to oxygen. As shown in Figure 5-9, our data suggested that the core
region of the fibrils is less accessible to oxygen comparing to the exposed N-
terminal. The less rigid sites in the core such as position 53, 81 are in fact more
accessible to oxygen comparing to other sites located within the core. Because
of the limitated available sites we could study, for now it is inconclusive which
sites are more surface-exposed and which sites are more buried within a ß-sheet.

R3 S61

S11 L65

K19 E69

F22 T73

S28 E77

S33 R81

D38 V85

E50 K91

D53 D96

S55

Figure 5- 8. Spectra of 25% R1-labeled ß2m LS fibrils (in pH 2.5, no salt).
All EPR spectra were obtained at room temperature using a 150 Gauss scan
width and were normalized to the same number of spins.

100
(0.05)
0.05
0.15
0.25
0.35
0.45
0.55
R3
S11
K19
F22
S28
S33
D38
E50
D53
S55
S61
L65
E69
T73
E77
R81
V85
K91
D96
Residue #
Mobility / Ox Accessibility
Mobility
Oxygen accessibility

Figure 5- 9. Plot of mobility and oxygen accessibility of 25% R1-labeled
ß2m LS fibrils (in pH 2.5, no salt).
Mobility (blue dots) or Oxygen accessibility (pink squares) was plotted as function
of the residue numbers.

Different Structure Presented in ß2m Worm-like (WL) Fibrils.
As we had introduced in the beginning of this chapter that under same
acidic condition, salt concentration played an important role in generating
different morphology types of fibrils. It was evident to observe the exchange
narrowing phenomena in the long-staright (LS) fibrils core region, however, it was
also clear that it was not the same case with the worm-like (WL) fibrils generated
in the same acidic condition of pH 2.5, but with 0.4M salt, as it presented in
Figure 5-10 below. Therefore, the data here suggested that worm-like (WL)

101
fibrils could take up a different structure comparing to the ordered parallel, in-
register structure owned by the long-straight (LS) forms. And more evidence is
required to make a definitive conclusion in this case.

102

K19 L65

F22 E69

S33 T73

D38 E77

D53 R81

S61 D96

Figure 5- 10. EPR Spectra of 100% R1-labeled ß2m WL fibrils (in pH 2.5, 0.4
M salt).
All EPR spectra were obtained at room temperature using a 150 Gauss scan
width and were normalized to the same number of spins.

103
5.5 Chapter 5 References
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Chiba, T., Hagihara, Y., Higurashi, T., Hasegawa, K., Naiki, H. and Goto, Y.
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106
CHAPTER 6:
CHARACTERIZATION OF SPIN EXCHANGE EFFECT USING A MODEL
PROTEIN D-AMPHIPHYSIN

6.1 Abstract

The single-line, exchange-narrowed EPR spectra phenomenon had been
observed in both α-synuclein fibrils and ß-2-microglobulin long-straight fibrils
systems, as well as shared by many other naturally occurring amyloid fibrils, here
we utilized a model protein D-amphiphysin to characterize the origin of spin
exchange. Our study have shown that in such simulated in vitro system, 4 spin
labels in close contact (about 5 Å proximity) with each other are sufficient to
cause the occurrence of spin exchange.

6.2 Introduction
Amphiphysins are a family of mammalian brain-enriched proteins involved
in synaptic vesicle endocytosis. They interact with clathrin, adaptor protein
complex 2 (AP2), dynamin and synaptojanin. Of all amphiphysins from yeast to
Drosophila and humans, the only conserved region is an N-terminal BAR
(Bin/amphiphysin/Rvs) domain, which is also present at endophilins and nadrins.
Recently the crystal structure of Drosophila amphiphysin Bar domain (D-
Amphiphysin, containing aa 1 - 245) was solved and shown an elongated
banana-shaped dimer (Peter et al. 2004). The N-terminal before aa 26 is
disordered as well as the C-terminal after aa 242. In the crystal structure, the
BAR domain runs from residues 35 to 240, where is the only homologous region
in this family of proteins. Each monomer is a three long kinked α-helices coiled-

107
coil; they form a six-helix bundle around the dimer interface that is mainly
hydrophobic. It is noted, in vivo, Amphiphysins are found to be in equilibrium of
monomer to dimer. In vitro and in vivo, Amphiphysins drive the membrane
curvatures with the help of its N-terminal amphipathic helix and BAR domain. D-
amphiphysins are found to be able to bind and tabulate liposomes both in vitro
and in vivo.
Here we took advantage of its dimeric structure and manually put in
different numbers of R1 labels at different distances to the protein and studied
the resulted EPR spectra to find out exactly how many spin labels and in what
close proximity spin exchange would take place.

6.3 Experimental Procedures
Generation of D-amphiphysin Cysteine Mutants
A Cys-less wild type D-amphiphysin (245aa) in His6 tag cloned in PET vector
was generously provided by colleague Dr. Mario Isas in the lab. Two native
cysteines exist in D-amphiphysin and they were converted to Alanines.
Cysteines were introduced by site-directed mutagenesis and verified by DNA
sequencing.

Protein Expression and Purification of D-amphiphysin Cysteine Mutants
Escherichia coli BL21(Gold)DE3 cells were transformed with the His6 PET
construct by heat shock. Expression of D-amphiphysin was induced with 1mM
isopropyl-1-thio-ß-D-galactopyranoside at 22 °C overnight. Cells were collected

108
by centrifugation at 4000 rpm for 20 min and lysed in resuspension buffer
containing 20 mM Hepes pH 7.0, 500 mM NaCl, 1 mM DTT, and 1 mM EDTA.
The cell lysate was then sonicated 3 times 5 min each time and followed by
centrifugation at 16,000 rpm for 25 min. The resulting supernatant was utilized to
purify the D-amphiphysin proteins through 3 different columns. The supernatant
was loaded onto a HiTrap HP Ni-NTA column (Amersham Biosciences, GE
Healthcare) equilibrated in the aforementioned resuspension buffer. The column
was washed using resuspension buffer containing additional 20 mM Imidazole
and the desired proteins were eluted in resuspension buffer containing additional
250 mM Imidazole. The eluted proteins were concentrated using Amicon Ultra-15
centrifugal filter units (MWCO 10×10
3
, Millipore) to around 5 ml and applied to
the Superdex200 gel filtration column (CV=300 ml) equilibrated in the same
resuspension buffer. The desired protein peak was eluted around 180-230ml. At
this point, the D-amphiphysin-containing fractions were pooled and diluted in the
buffer containing 20 mM Hepes pH 7.0, 1 mM DTT, and 1 mM EDTA, then
loaded onto a Mono-S HR5/5 column (Amersham Biosciences, GE Healthcare)
that was equilibrated with the same buffer. Proteins were then eluted in 0 - 2 M
NaCl gradient and fractions were analyzed by SDS-PAGE. Fractions containing
D-amphiphysin were subsequently pooled and 1 mM DTT was added. Protein
purity was > 95% according to Bio-Safe
TM
Coomassie Stain (Bio-Rad). Protein
concentration was determined by using a Micro BCA protein assay kit (Pierce).

Spin Labeling of D-amphiphysin Cysteine Mutants
Immediately before spin labeling, DTT was removed by loading the protein
solution onto a PD-10 column (Amersham Biosciences, GE Healthcare)
equilibrated with buffer containing 20 mM Hepes, pH 7.0, 500 mM NaCl and
1 mM EDTA, then eluted using the same buffer. D-amphiphysin was labeled with
10-fold molar excess of R1 MTSL spin label [1-Oxyl-2,2,5,5-tetramethyl-D-
pyrroline-3-methyl]-methanethiosulfonate (Toronto Research Chemicals, Toronto,
Ontario, Canada) or with a mixture of diluted R1 spin label and the diamagnetic
analog, R1’ [1-Acetyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl]-
methanethiosulfonate (Toronto Research Chemicals) (see Figure 6-1) for 1 hour
at room temperature. Excess label was removed using PD-10 columns with the
aforementioned elution buffer. Spin-labeled proteins were washed twice with
elution buffer, concentrated using Amicon Ultra-4 centrifugal filter units (MWCO
10×10
3
, Millipore).

Figure 6- 1. Chemical structure of the paramagnetic side chain R1 and its
diamagnetic analogue, R1’.
(Adapted from (Chen et al. 2007) ).

109

110
X-band EPR Spectroscopy and Data Analysis
The derivatized proteins were loaded into quartz capillaries (0.6 mm inner
diameter × 0.84 mm outer diameter, VitroCom, Mt. Lakes, NJ), and EPR spectra
were recorded on X-band Bruker EMX spectrometers (Bruker Instruments,
Billerica, MA). Full spectral scans were performed using an ER 4119HS
resonator at room temperature. The spectral scans obtained at room
temperature using the ER 4119HS resonator had a scan width of 300 Gauss at
an incident microwave power of 12 mW. All EPR spectra shown were
normalized to the same amount of spins, using double integration, and are
presented as normalized. For distance measurements, EPR spectra were
obtained at 233K using a Bruker N
2
temperature controller (ER4131VT), and
were recorded at 300 Gauss scan width. Simulation of dipolar broadening was
performed using software generously provided by Drs. Altenbach and Hubbell, as
described previously (Altenbach et al. 2001).

6.4 Results and Discussions
Exchange Narrowing Revealed in R1-labeled D-amphiphysin in Solution
According to D-amphiphysin dimer crystal structure (a snapshot is shown
in Figure 6-2, PDB code: 1URU, (Peter et al. 2004) ), the residue N220 is the
center site spaced at 7 Å from each other in the dimer, Q216 is spaced 5 Å away
from N220 on the α-helix, while A213 is spaced 10 Å away from site 220 on the
α-helix, and Q209 is spaced 10 Å away from Q216 on the α-helix. Because of

111
the unique position of N220, we therefore chose to start with it as our reference
site in our study.
Figure 6-3 showed spectra derived from R1-labeled D-amphiphysins we
had studied. As it shown in R1-labeled N220 derivatives, if we only put 2 labels
that are 5 Å from each other in the protein, we did not observe any exchange-
narrowed single line spectrum which suggesting 2 labels at close distance of 5 Å
is not sufficient to cause spin exchange. Subsequently, if we increase the
number of labels but keep the distance unchanged, such as here putting 4 labels
that remain 5 Å from each other, as it shown in R1-labeled Q216, N220
derivatives, we would be able to detect the exchange-narrowed single line
spectrum, which did not go away even at low temperature. This piece of data
suggested that the number of the labels are important to result in spin exchange,
in this case 4 labels that are at 5 Å from each other seemed be sufficient to lead
to spin exchange. Moreover, if these 4 labels that are spaced 5 Å from each
other are kept in the system, the additional labels added on to the system would
have slightly effect on the resulted spectra depending on how the tertiary
interactions are in the protein, as it displayed in the spectra from Q216, N220,
K224. In contrast, if we keep 4 labels but change the distances of the labels from
each other, such as in the example of A213, N220 in which the labels are
distanced at 10- 5 - 10 Å; or in the case of Q209, Q216 in which the labels are
apart at 10- 10 - 10 Å, we could not obtain any exchange-narrowed single line
spectra. Such data indicated that not only the number of labels played an

112
important role in the spin exchange, but also the distances of the labels from
each other.
Taken together from previous studies in α-synuclein, ß2m, IAPP and tau
fibrils (Der-Sarkissian et al. 2003; Jayasinghe et al. 2004; Margittai et al. 2004;
Chen et al. 2007), 5 Å is required to allow the orbital overlap of the unpaired
electrons and subsequently lead to spin exchange (Margittai et al. 2008) and 4
labels of these would be sufficient enough to cause it happen. Additional control
experiments and analysis would be required to draw a solid conclusion from this
study.

113

Figure 6- 2. A snapshot of D-amphiphysin dimer crystal structure.
The D-amphiphysin dimer is displayed in the line ribbon style (upper picture) and
in the Ca Wire style (lower picture), with pairs of residues 209, 213, 216, 220,
224 labeled in dimer. The distance of 220 - 220 is calculated around 5 Å, while
the distance of 216 – 220 is calculated around 4.87 Å.

114
Figure 6- 3. EPR spectra of 100% R1-labeled D-amphiphysin measured at
room temperature and frozen state.
Here show the EPR spectra of freshly prepared, predominantly dimeric spin-
labeled D-amphiphysin derivatives harboring one or more R1(s) at the indicated
positions. The reference site 220 is highlighted in red color, which is the center
site that spaced at 5 Å from each other in the dimer. The reference site 216 is
highlighted in blue color, 216 is spaced 5 Å away from site 220 on the α-helix.
The position 213 is spaced 10 Å away from site 220 on the α-helix. A) EPR
spectra shown in the panel highlights the importance of the number of the spin
labels for spin exchange; B) EPR spectra shown in the panel highlights the
importance of the distance of the spin labels to each other for spin exchange.
The EPR spectra shown at middle column were obtained at room temperature,
and the EPR spectra at right column were obtained at frozen state (233 K), all
spectra using a 300 Gauss scan width and were normalized to the same number
of spins. N/A represents not available.

Figure 6-3: Continued

A)
Room Temperature 233 K

N220 N/A

Q216, N220

Q216, N220, K224

B)
Room Temperature 233 K

Q216, N220

A213, N220

Q209, Q216

115

116
6.5 Chapter 6 References
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Chen, M., Margittai, M., Chen, J. and Langen, R. (2007). "Investigation of alpha-
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Abstract (if available)

Abstract This thesis discusses the application of site-directed spin labeling coupled with electron paramagnetic resonance spectroscopy in the structural analysis of amyloid fibrils derived from α-synuclein and ß-2-microglobulin.

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Asset Metadata

Creator Chen, Min (author)

Core Title Analysis of amyloid fibrils by site-directed spin labeling and electron paramagnetic resonance

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology

Degree Conferral Date 2009-08

Publication Date 07/29/2009

Defense Date 06/23/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag amyloid fibrils,electron paramagnetic resonance,OAI-PMH Harvest,structural analysis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Cadenas, Enrique (committee chair), Langen, Ralf (committee chair), Chen, Jeannie (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email chenmin@usc.edu,min.chen008@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2418

Unique identifier UC1496174

Identifier etd-Chen-2924 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-561145 (legacy record id),usctheses-m2418 (legacy record id)

Legacy Identifier etd-Chen-2924.pdf

Dmrecord 561145

Document Type Dissertation

Rights Chen, Min

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu