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Structural characterization of pseudorepeat shuffled alpha-synuclein

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Structural characterization of pseudorepeat shuffled alpha-synuclein

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STRUCTURAL CHARACTERIZATION

OF PSEUDOREPEAT SHUFFLED ALPHA-SYNUCLEIN

by

Leena S. Park

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2008

Copyright 2008 Leena S. Park

ii

Dedication

This work is dedicated to my family, who always supports me through all my life,

and to Dr. Ulmer and Dr. Rao who had given me countless advices.

iii

Acknowledgements

The protein had been prepared kindly by Dr. Ulmer, and NMR spectroscopy and
the structural calculations were also helped by Dr. Rao and Dr. Ulmer.

iv

Table of Contents

Dedication ii

Acknowledgements iii

List of Figures v

Abbreviations vi

Abstract vii

Introduction 1

Materials and Methods 10

Results 13

Discussion 21

Bibliography 25

v

List of Figures

Figure 1: Structures of SDS-bound aS 4
Figure 2: Sodium Lauroyl Sarcosine (SLAS) Structure 6
Figure 3: SDS-bound aS (de Laureto et al 2006) 7
Figure 4: Amino acid sequence of human alpha-synuclein (aS) 9
Figure 5: Amino acid sequence of shuffled alpha-synuclein (aSS) 10
Figure 6: TROSY of shuffled alpha-synuclein 14
Figure 7: Secondary Chemical Shifts of Wild-Type (black) and Shuffled (Red) 15
Figure 8: {1H}-15N Steady State NOE of Wild-Type aS (black) 17
and Shuffled aS(red) with the tail regions omitted due to the same
pattern of spectra.

Figure 9: Residue Dipolar NH Coupling of the Shuffled aS; 18
some blank areas are unassigned residues.

Figure 10: (a) Structure of Wild-Type SLAS-bound aS. 20
(b) Structure of Shuffled SLAS-bound aS.

vi

Abbreviations

AMPS 2-acrylamido-2-methyl-1-propanesulfonic acid
aS alpha-synuclein
aSS shuffled sequence of alpha-synuclein
CARA computer aided resonance assignment
CMC critical micelles concentration
HMQC heteronuclear multiple quantum coherence
HSQC heteronuclear single quantum coherence
MFR molecular fragment replacement
NMR nuclear resonance spectroscopy
NOE nuclear Overhauser Effect
RDC residual dipolar coupling
SDS sodium dodecyl sulfate
SLAS sodium lauroyl sarcosine
TROSY transverse relaxation-optimized spectroscopy

vii

Abstract

The 140-residue alpha-synuclein plays a central role in the molecular
chain of events leading to Parkinson’s disease. Alpha-synuclein is dynamically
unstructured in aqueous solution, but readily binds to negatively charged
membranes and micelles. Seven degenerate 11-residue repeats located within the
first 89 amino acid residues confer membrane binding in an alpha-helical
conformation. In this research, the structure of a pseudorepeat shuffled alpha-
synuclein variant is characterized by NMR spectroscopy and is compared to the
wild-type protein. It is shown that residues 30 to 40 become more rigid than the
corresponding wild-type segment. Moreover, in contrast to the wild type, which
prefers to adopt two anti-parallel helices, the shuffled variant prefers to adopt a
single helix.

1

Introduction

Synucleinopathies is the term used to describe the various types of
neurodegenerative disorders. They are characterized by the aggregates of alpha-synuclein
in the cytoplasm of neurons and glia (Goedert 1999; Spillantini et al 2000; Galvin et al
2001). Multiple system atrophy, dementia with Lewy bodies, and Parkinson’s disease are
the most famous and well-known synucleinopathies (Spillantini et al 2000).
Parkinson’s disease, one of the most common neurodegenerative diseases after
Alzheimer’s disease, is notorious for the symptoms that the patients can no longer
maintain everyday life. They develop motor dysfunction and psychiatric problems
(International Tremor Foundation 2008). More astonishingly, Parkinson’s disease, the
14
th
leading cause of death in the United States of America, increased the death rate about
5 percent in 2005 compared to 2004 (National Center for Health Statistics 2007). Not
only in the United States of America, it was predicted that Parkinson’s diseases will
increase double-fold in 15 biggest industrial countries in the world: the United States of
America will have 610,000 of the patients’ population and other 15 biggest countries will
also have the 8.7 million population by 2030. Parkinson’s disease causes enormous pain
to the patients themselves and their families, and it also brings severe social and
economic costs to the nation as well (Michaud 2007).
In Parkinson's disease, Lewy bodies and Lewy neuritis are found, and these
abnormal protein aggregations lead to cell death, thus finally cause serious
neurodegenerative diseases (Forster et al 1912; Pollanen et al 1993; Kuzuhara et al 1988).
Several experiments proved that alpha-synuclein is a major component of Lewy bodies

2
(Spillantini et al 1997; Mezey et al 1998). Additionally, the protein is able to form
fibrils, which eventually becomes the main cause of the neurodegenerative disease, and
its tendency to form fibrils depends on two main factors. First, the fibril formation of the
protein can be accelerated by environmental factors, such as pesticides/herbicides
(Vanacore et al 2002), numerous metal ions (Uversky et al 2001), organic solvents
(Munishkina et al 2003), and phosphorylation (Fujiwara et al 2002). Numerous pesticides
and herbicides used in the United States increased the prevalence rate of Parkinson’s
disease. Also, mono-, di-, trivalent metal ions increased the tendency of alpha-synuclein
fibrillation (Uversky et al 2001). Organic solvents at low concentration are other factors
that enhance the partial folded conformation of alpha-synuclein that is prone to form
fibril (Munishkina et al 2003). Lastly, when alpha-synuclein, normally non-
phosphorylated in vivo, is phosphorylated at Ser129, it enhances formation of oligomers
and fibrils, which are widely found in alpha-synucleinopathy lesions and in aged human
brains (Fujiwara et al 2002).
Genetic factor is again another factor to lead the disease; and, several experiments
showed the evidence that three missense point-mutations, Ala53Thr in Italian-American
family (Polymeropoulos et al 1997), Ala30Pro in German family (Krüger et al 1998), and
Glu46Leu in Spanish Family (Zarranz et al 2004) are the most common genetic
alterations in familial Parkinson’s disease. These point mutations tend do increase alpha-
synuclein aggregate in vitro (Conway et al 2000). In addition, gene triplication of alpha-
synuclein causes familial Parkinson’s disease as well, showing that over-expression of
alpha-synuclein can cause the early-onset of Parkinson’s disease (Singleton et al 2003).

3
Alpha-synuclein is found to be highly conserved in different organisms
(Clayton and George 1998; Beyer 2006). There are at least three different isoforms in
humans, generated by splicing (Clayton and George 1998; Beyer 2006). The best-
identified isoform is alpha-synuclein-140, and it had been used here, too.
Alpha-synuclein is a natively unfolded protein in physiological conditions both in
vitro and in vivo; thus, it does not take any unique structures (Uversky et al 2001;
Weinreb et al 1996; McNulty et al 2006). However, the protein can change its
conformation depending on various environments.
The protein can take the pre-molten globule state at various conditions, such as
low pH, high temperature, organic solvents at low concentrations, etc (Munishkina et al
2003; Uversky et al 2001). Also, with negatively charged small unilamellar vesicles or
detergent micelles present, the protein forms alpha-helical membrane bound
conformation at the N-terminal fragments (repeat regions).
Later, it was found that, with SDS-micelles bound, the protein forms an
interrupted break between two helices (Davidson et al 1998; Ulmer et al 2005; Eliezer et
al 2001) (Figure 1). Val3-Val37 (helix-N) and Lys45-Thr92 (helix-C) form alpha-helices
in anti-parallel arrangement, connected by a well-ordered extended linker which is
appeared to be a special feature of the micelle-bound structure (Ulmer et al 2005). Then,
the protein has a short extended stretch (Gly93-Lys97), followed by a tail region. The fact
that the short extended stretch, Gly93-Lys97, overlaps with the chaperone-mediated
autophagy recognition motif of alpha-synuclein (Val95-Gln99) may show a good
evidence of damaged alpha-synuclein degradation in Parkinson’s disease (Ulmer et al
2005). The C-terminal region of the protein is a highly mobile tail and remains

4
unassociated even when vesicles and micelles are present (Ulmer et al 2005; Eliezer et
al 2001). The protein’s association with small unilamellar vesicles and micelles supports
the evidence that alpha-synuclein is involved in presynaptic vesicles in vivo and it is
localized mostly at axon termini (Iwai et al 1995; Jensen et al 1998)

Figure 1. Structures of SDS-bound aS: 20 different SDS-bound aS is being
superimposed while C-terminal random coil regions vary (Ulmer et al 2005)

Other than forming an alpha-helix, alpha-synuclein can also form beta structural
species with the addition of various alcohols. Simple alcohols increase the tendency of
beta-sheet formation and beta-structure rich conformations, and they also amplify the
formation of amorphous aggregates (Munishkina et al 2003).
Alpha-synuclein is, moreover, able to form dimers when incubated at high
temperatures (Uversky et al 2001). The protein can also form oligomers, too. When
alpha-synuclein is incubated with different metals, it induces three different sized
oligomers (Lowe et al 2004). An earliest form of alpha-synuclein protofibrils is spherical

5
and later it is converted to rings (Conway et al 2000; Ding et al 2002). Finally, alpha-
synuclein forms amorphous aggregates and fibrils, based on the condition of the solution.
At high concentrations of simple alcohols, it forms amorphous aggregates, while other
insoluble species are mainly amyloid-like fibrils (Uversky 2007). In conclusion, alpha-
synuclein, a natively unfolded protein, can take morphologically different forms of
conformations, showing that its great plasticity is another explanation that the protein is
potentially prone to misfold and develop synucleinopathies (Uversky 2007).
Partial folding or misfolding of alpha-synuclein into the fibrillation-prone
conformation is a key intermediate pathway in the fibrillation, and it is found that alpha-
synuclein oligomers are neurotoxic (Uversky et al 2001). In the patients with gene
triplication mutation, oligomeric alpha-synucleins are deposited in detergent insoluble
forms (Miller et al 2004). Also, the fact that the Ala30Pro mutant stimulates the initial
oligomerization provides good evidence for the relation between oligomeric toxicity and
the disease (Conway et al 2000; Li et al 2002; Li et al 2001). Besides, intermediate fibril
or oligomer is responsible for neuronal death, indicating strong evidence that
neurodegenerative diseases are caused by the formation of protofibrillar species as well
(Goldberg and Lansbury 2000).
For this research, natively unfolded alpha-synuclein is bound to sodium lauroyl
sarcosine (SLAS) (Figure 2.) to induce helical formation. When the protein is bound to
synaptic vesicles, it forms elongated and amphiphilic helices (Davidson et al 1998; Jao et
al 2004). Since such lipoproteins do not fold without vesicles, micelles or vesicles need to
be introduced in vitro to form the folded structures of the proteins (Davidson et al 1998).
Although a vesicle may be a good candidate, a vesicle with large size prevents NMR

6
spectroscopy to acquire a complete structure of a protein. Instead, detergent molecules
are used to mimic the vesicle effects. Commonly used detergent molecules imitate
membranes well enough, but its structure and plasticity may restrict the length of
amphiphilic helices of micelles, thus, lead to the local alteration of a helix such as helix
curvature (Chou et al 2002).
For example, with sodium dodecyl sulfate (SDS) or lysophospholipid micelles,
the amphiphilic regions of alpha-synuclein form two anti-parallel helices (Ulmer and Bax
2005), as shown on Figure 3. However, this may be less likely to happen with synaptic
vesicles because they are 10 times bigger than SDS micelles (Kim et al 2000). SLAS has
much bigger aggregation number, thus, bigger micelle size, compared to SDS (Table 1).
Indeed, among several different types of detergent tested, SLAS had been found as a
most ideal detergent for NMR analysis of alpha-synuclein because it showed the most
suitable interaction between the protein and the detergents. Additionally, by using SLAS,
the resonance detection fell in good range, so the spectra were obtained in high quality
for both below and above its CMC (~14mM) (Rao and Ulmer 2008; Sehgal et al 2006).
Therefore, SLAS had been used in this experiment to mimic the vesicle effects.

Figure 2. Sodium Lauroyl Sarcosine (SLAS) Structure

7

Figure 3. SDS-bound aS (de Laureto et al 2006)

Detergent Type CMC (mM) Aggregate Number Micelle Molecular
Weight (g/mol)
SDS
(Sodium dodecyl
sulfate)
7-10 62 17879.56
SLAS
(Sodium lauroyl
sarcosine)
14.6 104 30600

Table 1. Comparison between SDS and SLAS (Wikipedia 2008; Sigma Aldrich 2008)

NMR spectroscopy is used to obtain the structural data of alpha-synuclein. The
structures of small proteins and DNA fragments are determined by the NMR
spectroscopy. Indeed, not all proteins are available for X-ray crystallography because
some are hard to crystallize. To get NMR spectra, a sample is placed on a homogeneous
magnetic field and electromagnetic energy is applied at certain frequencies. When

8
determining the macromolecules like proteins, there are mainly two big problems. First,
the signals are overlapped due to the large number of resonances. The second problem is
that the larger molecules relax faster. These two problems make difficult to analyze the
spectra. However, transverse relaxation-optimized spectroscopy (TROSY) made greater
possibilities in NMR application to macromolecular structures by reducing transverse
relaxation (Fernandez and Wider 2003), and it was used in this experiment, too.
Doubtlessly, there are other different types of data in obtaining the structural
information from NMR, but some of the mainly used spectra are only briefly explained
here. First, to correlate different nuclei, HMQC (heteronuclear multiple quantum
coherence) and HSQC (heteronuclear single quantum coherence) are used (Oschkinat
1994). NOE, nuclear Overhauser Effect, determines the neighboring nuclei through a
space by cross relaxation (Oschkinat 1994), thereby, giving out the distance information.
Also, scalar or J coupling measures the information through a bond, providing local
dihedral angle measurements, based on a phenomenon that nearby nuclei also affect the
nuclear frequency. In scalar coupling, it may be hard to detect overlapped residues. On
the contrary, dipolar coupling (residual dipolar coupling or RDC) reads the relative
angles of a number of bond vectors. However, dipolar coupling is not related to the
distance of atoms. It only depends on the relative orientation with respect to a common
axis system. In NMR analysis for protein structures, dipolar coupling is used to refine a
structure further which was determined by J coupling and NOE in advance. Summing up,
NOE and scalar coupling only evaluates the structure information between close atoms
while RDC provides more wide-scale orientations between remote internuclear vectors.

9
Therefore, putting these spectra together will give more accurate and more detailed 3D
protein structures (Kurt 1996).
The aim of this research is to look for any difference in structures after shuffling
imperfect repeat regions in the protein, using data obtained from NMR spectroscopy.
The wild-type sequence of alpha-synuclein is shown on Figure 4. There are seven
imperfect 11 repeats (KTKEGV) in the region between Ser9 and Ala89, followed by a
tail region, and figure 5 shows the shuffled sequence of alpha-synuclein. It has been
shuffled around so that the flexible regions of the wild-type replaced to the rigid regions
or vice versa. Roman numerals shown on each imperfect repeat above alpha-synuclein
sequences appear on the shuffled alpha-synuclein, too, in order to show how the region
has been shuffled. Then, using NMR spectroscopy, any difference between the wild-type
and the shuffled had been detected.

1 9 - I 20 - II 31 - III
MDVFMKGL SKAKEGVVAAA EKTKQGVAEAA GKTKEGVLYVG

42 – IV 53 57 - V 68 - VI 79 - VII
SKTKEGVVHGV ATVA EKTKEQVTNVG GAVVTGVTAVA QKTVEGAGSIA

90 100 110 120 130 140
AATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA
Figure 4. Amino acid sequence of human aS (excerpted from Ulmer et al 2005)

10

1 9 - V 20 - IV 31 - VI
MDVFMKGL EKTKEQVTNVG SKTKEGVVHGV GAVVTGVTAVA
42 - II 53 57 - I 68 - III 79-VII
EKTKQGVAEAA ATVA SKAKEGVVAAA GKTKEGVLYVG QKTVEGAGSIA
90 100 110 120 130 140
AATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA
Figure 5. Amino acid sequence of aSS

Further understanding of the alpha-synuclein structures will uncover the
neuropathology of the protein.
Materials and Methods
Protein Production Shuffled alpha-synuclein protein was purified and produced
by Dr. Tobias Ulmer.
NMR Sample Preparation In water, each sample consisted of aSS at 0.5 mM
(ε
280
=5120 M
-1
cm
-1
), 15 mM SLS, 6% deuterium oxide, and 0.02% (w/v) NaN
3
and total
volume added up to 270 μl. Total 4 samples were prepared. Sample A contained only
15
N.
Sample B was evenly distributed with
2
H/
13
C/
15
N. Sample C and D were
2
H/
13
C/
15
N with
aSS with charged stretched polyacrylamide gels. All samples used the buffer 20 mM
NaH
2
PO
4
/Na
2
HPO
4
at pH 7.4.
NMR Gel Preparation First of all, 2.5% bis+5.0% 2-acrylamido-2-methyl-1-
propanesulfonic acid (AMPS) stock solution had been made with 40% acrylamide, 10 ml
of 40% AMPS in 150 mM TrisHCl at pH 8.0 adjusted by NaOH tablets, 5 ml of 2% bis,

11
250 ml of 150 mM TrisHCl at pH 8.0. The ratio of acrylamide to bis (w/w) was 1:39.
Specificallym, 2.084 l of 40% acrylamide, 1.125 ml of 2% bis, 0.320 ml of 40% AMPS,
and 2.471 ml of 150 mM TrisHCl at pH 8.0 were used to make total 6 ml volume of stock
solution.
To make 5.2% of 330 μl NMR gel, 6.6% v/v TEMED (4 μl TEMED in 56.0 μl
150 mM TrisHCl buffer) and 6.6% w/v ammonium persulfate (APS) in 150 mM TrisHCl
buffer were freshly prepared (approximately 0.01 g of APS in 15 times of weight of 150
mM TrisHCl buffer). First, 195.6 μl of 150 mM TrisHCl buffer was added to a 1.5 ml
eppendorf tube. Then, 114.4 μl of gel stock solution that had been prepared before was
added. 10 μl of 6.6% v/v TEMED was added next and 10 μl of 6.6% w/v APS was added
lastly because APS would start polymerization immediately. These four solutions were
mixed up well and 320 μl of mixed solutions were transferred to 6.0 mm cylinder
instantly. Then, 320 μl of butanol was added over the solutions and polymerization had
been waited for 1 hour. 10 μl l leftover of solutions could be the measure to check the gel
was polymerized well.
Once the gel was polymerized, the gel needed to be soaked in 50 ml of 100 mM
NaP at pH 6.8 overnight. Next morning, 50 ml of 100 mM NaP at pH 6.8 had been
replaced to 50 ml of MilliQ-water, and it needed to be changed again in evening.
From next day, the gel was to be dried. After the gel was placed onto the
weighing boat carefully, the gel was put under the 37°C incubator for whole day, and the
gel was loosened with MilliQ-water. Then, remained water was soaked up by KimWipe
tissue, avoiding touching the gel. The gel was put under the sterile hood overnight. Next
morning, the gel was placed in 37°C incubator. Every 30-60 minutes, the gel was neede

12
to be loosened up. Touching the gel with the tissue or the gel sticking to the weighing
boat should be avoided because it would damage the gel critically.
Next step was soaking the gel into the protein +detergent solution. To 1.5 ml
eppendorf tube, total volume of 320 μl protein and detergent solution were added (15 mM
SLS, 0.5 mM aSS, D2O, 20 mM NaP) and the gel was soaked into the tube for two
complete days.
20-30 μl of detergent only solution was applied to the eppendorf tube to prevent
gel rupturing. The cylinder was also wet with 10 μl of detergent only solution and the gel
was inserted into the cylinder. Thicker end always went first than the thinner end. The
transfer funnel and open-ended NMR tube had been wetted with 10 μl each of detergent
only solution and the gel was transferred into the tube now. Then, another 10 μl of
detergent only solution was applied to the bottom side of the gel, and Shigemi plunger
was inserted until the gel reaches the bottom plug. Keeping the NMR tube vertically, it
had been kept for overnight at room temperature.
NMR Spectroscopy All experiments were performed at 25°C on Bruker
spectrometers at 700 MHz of
1
H frequencies under cryogenic temperature. NMRPipe
package was used to process and analyzed the data (41). Also, the TROSY N-H
component was picked for the experiments (42, 43). HNCA, HNCB, HNCACO, and
HNCO experiments had been done (22).
R
1
, R
2
, and {
1
H}-
15
N NOE, the
15
N relaxation parameters were experimented at
60.8 MHz for sample B (48, 49). To measure {
1
H}-
15
N NOE, 5 s of presaturation and a
4s of recycle delay were used for the NOE experiment and a 9 s of recycle delay were
used for the reference experiment, instead.

13
Secondary Structure Calculation Since C
α
and C΄resonate down in alpha-
helix and they resonate up in beta-sheets, the secondary structure of the protein could be
predicted (Wishart and Sykes 1994). Based on the random coil shift values (Wishart and
Sykes 1994; Wishart et al 1992), the secondary chemical shifts were calculated for the
protein.
Structure Calculation On CARA using HNCA, HNCB, HNCACO and HNCO
spectra, each residue had been assigned.
Residual dipolar coupling was tested under a weak alignment of the protein. It
was obtained by getting a difference between splitting of partially aligned and isotropic
spectra (Prestegarda et al 2005).
Molecular fragment replacement (MFR) is also useful when obtaining backbone
structure of proteins (Kontaxis et al 2005). MFR is based on RDC measurements, and it
is best suited for proteins rich in alpha-helix (Wu et al 2005). MFR looks for
substructures in the Protein Data Bank (PDB) that are well-matched with dipolar
couplings and chemical shifts already measured for a given fragment of the target protein
(Kontaxis et al 2005).
Results
Figure 6 shows the TROSY of shuffled alpha-synuclein. The number has been
assigned next to the corresponding residues. However, the residue number 44 and 140 are
not clearly shown, so they are not indicated here.

14

Figure 6. TROSY of shuffled alpha-synuclein

15

Figure 7. Secondary Chemical Shifts of Wild-Type (black) and Shuffled (Red)

16
For this research, NMR analysis is used extensively. NMR analysis is a powerful
tool in determining secondary structures without obtaining 3D structures because it can
localize the secondary structures to the specific polypeptide chains while circular
dichroism spectroscopy (CD) or infrared spectra provides information only about overall
contents of the secondary structures (Berndt 1996).
As seen in the Cα chemical shifts graph on Figure 7, first 100 residues (more
specifically 3-90) are folded to alpha-helix. Typically, secondary Cα chemical shifts for
alpha-helix are higher than those of random coil regions. The tail regions of the wild-type
and the shuffled are same because the tail regions had not been shuffled.
To show the structural information about a space such as the internal flexibility of
the protein backbone, [
1
H]-
15
N NOE had been measured and shown in Figure 8. The
shuffled sequence has been re-ordered to the original position again to compare to the
imperfect repeat regions of wild-type alpha-synuclein. First imperfect repeat (I in Figure
4) mostly shows more flexibility when compared to the wild-type. Region II also shows
more rigidity; and, 8 out of 11 residues are more rigid while 3 out of 11 are more flexible.
In region III, last 2 residues show almost same degree of flexibility while first 9 show
more rigidity. It is hard to predict the difference of rigidity between wild-type and
shuffled sequence in region IV because some residues from wild-type protein had been
missed. However, some of the shuffled sequence definitely demonstrates more rigidity.
Lastly, region V and VI, displays that half of residues are more rigid and another half are
more flexible. Overall, the shuffled sequence of the protein appears more flexible.
However, the tail regions are not shown on the graph because those had not been shuffled
around.

17
N-H dipolar coupling constants are shown on Figure. 9. The graph supports the
data obtained from other graphs because it also shows the uniform patterns in alpha-helix
region and in random coil tail regions.

Figure 8. {1H}-15N Steady State NOE of Wild-Type aS (black) and Shuffled aS(red)
with the tail regions omitted due to the same pattern of spectra.

18

Figure 9. Residue Dipolar NH Coupling of the Shuffled aS; some blank areas are
unassigned residues.

19
Finally, the preliminary 3-D structure of the wild-type protein is shown on
Figure.10a as that of the shuffled protein is shown on Figure.10b. While wild type has
two alpha-helices connected by the partially folded regions, shuffled sequence has only 1
single uninterrupted alpha-helix. Random coil regions are found in both structures.
Additionally, in the shuffled protein, there appears to have two kinks in helical regions
(Figure. 10b).

20
(a)

(b)

Figure 10. (a) Structure of Wild-Type SLAS-bound aS. (b) Structure of Shuffled SLAS-
bound aS.

21

Discussion
Based on the results, the protein forms only alpha-helix as secondary. Thus, it
shows that the shuffled sequence, like the wild-type, is still forming alpha-helix only at
N-terminal and the random coil at C-terminal, with SLAS present. Also, the values of the
random coil regions of the shuffled protein mostly are as same as those of the wild-type
sequence, which indicates that the random coil regions are not affected by shuffling the
sequences. Therefore, it confirms that only the repeat regions are adjusting their
conformations to environments while the random coil regions remain unchanged.
Overall, the shuffled alpha-synuclein indicates more rigidity in helical regions
compared to wild-type, especially between the residue numbers 26 to 39. This region
(mostly the part of region III in figure 4), had been shuffled to the very end of the helical
regions where located right before the C-terminal tail regions start. C-terminal regions
had been shown performing a critical role in protofibrillation (Qin et al 2007). However,
the random coil regions (as well as the repeat regions) are not involved in the fibrillation,
which is the highest hierarchy of fibril that leads to actual disease (Tashiro et al 2008).
Rather, the initial fibrillation may be initiated by 7 imperfect repeats, especially the core
regions of about 70 amino acids (K32-K102) where contains the β-rich core of the
protofilaments and fibrils (Tashiro et al 2008; Gao et al 2007). In addition, these regions
(specifically E36-D98) are tightly packed in parallel of alpha-synuclein fibrils (Chen et al
2007).
Increased rigidity of region III after it has been shuffled to the very last region of
the imperfect repeat regions shows that this region may be important in initiation of

22
alpha-synuclein misfolding in wild-type, given that the Ala30Pro point-mutation of
familial Parkinson’s disease takes place in this area. Ala30Pro mutation disturbs the
structure and dynamics of the wild-type protein, and it also destabilizes helix-N. Also, the
residue numbers from 3 to 37 show lower helical characteristics than helix-C or other
residues of helix-N (Ulmer and Bax 2005). In other words, the region III in the wild-type
protein, is more flexible than any other regions and interacting with the detergents
molecules more weakly to form a partially folded structure, indicating further that the
decreased rigidity of conformation may lead to the initial stage of alpha-synuclein
misfolding (Ulmer et al 2005).
Dipolar N-H couplings shown on Figure 7 had been used to refine a structure
further by acquiring the global orientation between distant molecules. Since each region
(alpha-helical and random coil) follows each own pattern, it supports the structural idea
about the helical and the tail regions. Additionally, the alternating patterns between
positive and negative values around 100 residues show that the region is partially
unfolded structure since it is the in-between regions from the helical to random coil.
The preliminary structures of the wild-type protein and the shuffled sequence
protein do not show identical forms. The most remarkable point here is that the partially
folded region around 30 to 40 which was present in the wild-type protein is not found in
the structure of the shuffled sequence, which was found as a more extended form in the
SDS-bound protein (Rao and Ulmer 2008). SLAS had been used here because it was
hypothesized that it would show an uninterrupted helix without any partially folded
regions by mimicking the synaptic vesicles. However, the preliminary structure of the
SLAS-bound wild-type protein shows the partially folded region, although the region

23
takes more folded form than the SDS-bound protein. Additionally, since the partially
folded region becomes more folded in the shuffled SLAS-bound protein, it means the
regions became more rigid. The fact that the region around the residue 30 to 40 is
partially folded in the SLAS-bound wild type micelles may show that this region is more
susceptible to the environmental change than any other regions. Therefore, missing this
region in the shuffled sequence, it may indicate that it lost the favorable binding to the
detergent molecules, and losing the favorable interaction of this region may also decrease
the tendency of fibril formation. However, this needs to be tested further.
Thus, it was concluded that around residue numbers 30 to 40 showed less
interaction with the detergent molecules than other residues, which means weaker
interaction with the detergents, and maybe with the vesicles, too, in the body.
Further investigations need to be made for understanding alpha-synuclein’s
structure and behavior. Since the partially folded regions changed its dynamics mostly
among the residues, it is necessary to examine its direct role in fibril formation. Also, if it
is possible, it is good to know the definite roles of each imperfect repeat in fibril
formation. Another study can be done by testing the difference between alpha-synuclein
and other synuclein families. Additionally, the content of Gly is fairly high in alpha-
synuclein as globular proteins and integral membrane proteins, indicating that Gly
residues in the protein may have a functional role in dynamics. From the study, it was
predicted that Gly residues contribute to increasing membrane fluidity (Ulmer et al 2005).
Thus, investigating further how these Gly residues participate in the protein will help to
explain the dynamics of the protein. Putting these altogether, it is necessary to understand
the minute pathways of fibril formation of the protein.

24
Our final goal is to find the cure of Parkinson’s disease and other
synucleinopathies. Understanding the mechanism of Parkinson’s disease fully will lead
the way to cure other neurodegenerative diseases.

25
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Abstract (if available)

Abstract The 140-residue alpha-synuclein plays a central role in the molecular chain of events leading to Parkinson' s disease. Alpha-synuclein is dynamically unstructured in aqueous solution, but readily binds to negatively charged membranes and micelles. Seven degenerate 11-residue repeats located within the first 89 amino acid residues confer membrane binding in an alpha-helical conformation. In this research, the structure of a pseudorepeat shuffled alpha-synuclein variant is characterized by NMR spectroscopy and is compared to the wild-type protein. It is shown that residues 30 to 40 become more rigid than the corresponding wild-type segment. Moreover, in contrast to the wild type, which prefers to adopt two anti-parallel helices, the shuffled variant prefers to adopt a single helix.

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Asset Metadata

Creator Park, Leena S. (author)

Core Title Structural characterization of pseudorepeat shuffled alpha-synuclein

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2008-08

Publication Date 06/27/2010

Defense Date 03/25/2008

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

Tag alpha-synuclein,OAI-PMH Harvest

Language English

Advisor Tokes, Zoltan A. (committee chair), Langen, Ralf (committee member), Ulmer, Tobias (committee member)

Creator Email psy3572@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1299

Unique identifier UC1101712

Identifier etd-Park-20080627 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-80230 (legacy record id),usctheses-m1299 (legacy record id)

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Document Type Thesis

Rights Park, Leena S.

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