University of Southern California Dissertations and Theses

Glycine to alanine mutations affect the structure and dynamics of micelle bound alpha-synuclein

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Glycine to alanine mutations affect the structure and dynamics of micelle bound alpha-synuclein

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Content GLYCINE TO ALANINE MUTATIONS AFFECT THE STRUCTURE
AND DYNAMICS OF MICELLE BOUND α-SYNUCLEIN

by
Adithya Balasubramanian

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 Adithya Balasubramanian

ii

DEDICATION
Dedicated to my grandparents; Late.Mr.G.K.Ramasamy, Mrs.Savitri Ramasamy,
Mr.R.Swaminathan and Late.Mrs.Kamla Swaminathan.
iii

ACKNOWLEDGEMENTS
This work would not have been possible without the support and encouragement of my
advisor Dr.Tobias S. Ulmer. I would also like to thank my Thesis Committee for taking
their time to complete this work. I would also like to thank Dr. Nageshwara Rao Jampani
who constantly helped me out to finish this work without whom this research would have
been most difficult for me to complete. I would also like to thank Dr. Crystal and Varun
Dua for helping me out in various techniques in the laboratory.
I cannot end without thanking my parents and family, on whose constant encouragement;
I have relied throughout my time in this country. I am grateful also to the examples of my
father Mr. G.R.Balasubramanian and my uncle Dr.J.S.Bhuvaneswaran who have
supported me and given constant encouragement. Their hard-work and humbleness will
always inspire me. I would also like to thank my uncle Mr. Nandy Kumar for his constant
help and support.
I would also like to thank Chris Martin, Richard Ashcroft and A.R.Rahman for their good
music which kept me awake and gave me a great company.
Last but not least I would like to thank all my roommates and friends for all the good
times we have had at 2148 Oak Street.

iv

TABLE OF CONTENTS
Dedication ii

Acknowledgments iii

List of Figures vi

Abbreviations vii

Abstract viii

Chapter 1: Introduction 1
1.1 What is Parkinson’s disease? 1
1.2 α-synucleins (aS) 2
1.3 Structural features of aS 2
1.4 Function of aS 5

Chapter 2: Background and aim of the study 6

Chapter 3: Materials and Methods 9
3.1 Production of aS variant constructs 9
3.2 Expression of the aS variants 10
3.2.1 Expression of unlabelled aS variants in
Luria-Bertani (LB) media 10
3.2.2 Expression of
15
N labeled aS variants in
M9 Minimal medium 11
3.2.3 Expression of
13
C,
15
N labeled aS variants in
M9 Minimal media with D
2
O 12
3.3 Cell lysis 13
3.4 Purification and Characterization of aS 14
3.4.1 Cation exchange chromatography 14
3.4.2 Concentration of the aS protein 15
3.4.3 Size exclusion chromatography 15
3.5 Buffer exchange of aS proteins 16
3.6 Determination of protein concentration 17
3.7 SDS-PAGE electrophoresis 17
3.8. NMR spectroscopy 18
3.8.1 NMR sample preparation 18
3.8.2 NMR spectrometers and measurement 19
3.8.3 NMR data processing and analysis 20
3.8.4 Secondary structure determination by
NMR spectroscopy 20

v

3.8.5 Distance restraint analysis using NMR
Spectroscopy
1
H-
15
N heteronuclear NOE 21

Chapter 4: Results 23
4.1 Construction of the aS mutants, aSG(III) and aSG(II) 23
4.2 Expression and purification of recombinant aS variants 23
4.3 Cation exchange chromatography 24
4.4 Size exclusion chromatography 28
4.5
1
H-
15
N TROSY of aSG(II) and aSG(III) 30
4.6 Normalized chemical shift changes 31
4.7 C
α
chemical shifts 36
4.8
1
H-
15
N Heteronuclear NOE 39

Chapter 5: Discussion 42

Chapter 6: Conclusion 45

References 46

vi

LIST OF FIGURES
Figure 1: Model of α-Synuclein aggregation. 3
Figure 2: Amino acid sequence of α-Synuclein 4
Figure 3: Structure of micelle bound α-Synuclein 7
Figure 4: Chemical Structure of SLS 7
Figure 5: Amino acid sequence of the mutants 25
Figure 6: Chromatogram of Cation Exchange chromatography 26
Figure 7: Chromatogram of Size Exclusion chromatography 27
Figure 8: SDS-Gel picture 29
Figure 9: TROSY overlay of aSG(III) with aS(wt) 32
Figure 10: TROSY overlay of aSG(II) with aS(wt) 33
Figure 11: Normalized weighted chemical shift changes for aSG(III) 34
Figure 12: Normalized weighted chemical shift changes for aSG(II) 35
Figure 13: C
α
chemical shift changes of aSG(III) 37
Figure 14: C
α
chemical shift changes of aSG(II) 38
Figure 15: Het-NOE of aSG(III) 40
Figure 16: Het-NOE of aSG(II) 41

vii

ABBREVIATIONS
PD Parkinson’s disease
aS α-Synuclein
SUV Small Unilammelar Vesicles
SNARE Soluble N-ethylmaleimide sensitive fusion protein attachment
receptor

NMR Nuclear Magnetic Resonance
TROSY Transversed Relaxation Optimized Spectroscopy
SLS Sodium lauroyl sarcosine
SDS Sodium dodecyl sulfate
PCR Polymerase Chain Reaction
PAGE Polyacryl amide gel electrophoresis
MWCO Molecular weight cut-off
NOE Nuclear Overhauser effect
Het-NOE Heteronuclear Nuclear Overhauser effect
CMC Critical Micelle Concentration
viii

ABSTRACT
Parkinson’s disease has been associated with misfolding of the protein α-synuclein (aS).
Previous studies of micelle bound α-synucleins have shown that it forms two anti-parallel
helices on the micelle surface with elevated dynamics in the glycine residues of the III,
V, and VI repeat of its amino acid sequence. Thus by mutating these residues to alanine a
significant change in the dynamics and structure has been observed. A better micelle
system has been established using sodium lauroyl sarcosine (SLS) which has more
aggregation number than SDS and decreases the negative restraint on the C
α
chemical
shifts. Two aS variants, aSG(III) with mutated Gly residues at 31, 36 and 41 mutated to
Ala and aSG(II) with mutated Gly residues at 67 and 68 to Ala are studied. Backbone
and dynamic parameters of the variants show that there is an increase in chemical shift
causing an increment in helical character in the regions of mutation. But this is been
compensated at other distant residues where the same character is significantly reduced.
Similar pattern is observed in the dynamic parameters which show an increase in rigidity
of the helix in the mutated regions. These strong increase in chemical shift and decrease
in dynamics suggest that the glycine residues in these positions play a significant role in
the interaction of the protein α-synuclein with the lipid surfaces.
1

1. INTRODUCTION:
1.1 What is Parkinson’s disease?
Parkinson’s disease (PD) is a degenerative disorder of the central nervous system.
The National Institute of Health (NIH) describes that PD belongs to group of conditions
called motor system disorders.
PD is one of the most common neurodegenerative diseases, although, until
recently little was known about its aetiology and the molecular composition of its
defining neuropathological characteristic the Lewy Body (34). Later stages of PD are
characterized by loss of neurons leading to the suppression of motor neurons ending in
death. Lewy bodies that are found within the neurons are deposits of protein compounds
along with other elements like lipids and neurofilaments (39). These thread-like
proteinaceous inclusions called Lewy neurites have also been identified as a
characteristic feature of PD (39). It is identified that the deposits in Lewy bodies and
Lewy neurites are a 140-residue protein compound called α-synuclein (28, 40). Along
with α-synucleins other components like neurofilaments and cytoskeleton elements are
present (18).
Three missense mutations (A30P, E46K and A53T) in the aS gene were found to
cause the familial form of PD (35). Lewy bodies that are present in the cases of sporadic
PD were found to be strongly immunoreactive for aS (40). This confirmed that the
filamentous inclusions of the Lewy bodies were made of aS. Increased cellular
accumulation of aS also leads to the symptoms of PD.

2

1.2 α 1.2 α 1.2 α 1.2 α-synucleins (aS):
Synucleins are proteins that are abundant in the brain which are of three types; α-
synuclein, β-synuclein and γ-synuclein. Physiological functions of them are poorly
understood. Misfolding of aS is caused from oxidation of aS by oxidative stress and
reactive oxygen species, ROS (Fig.1) created by impairments in mitochondrial complex-I
activity (5, 9).
Many recent studies support a contrary fact that the aggregation of these proteins
are actually neuroprotective, compensatory responses mounted by neurons against
neuroprotective stress (27). For example the oxidative stress caused by the herbicide
paraquat causes aS aggregation in brains of experimental animals (27). Inactivation of aS
gene by homologous recombination does not lead to a severe neurological phenotype. So,
loss of function of the aS protein is unlikely to account for its role in neurodegeneration
(1).

1.3 Structural features of aS:
The aS family consists of three distinct genes, α-synuclein, β-synuclein and γ-
synuclein which have been described for vertebrates only. The amino acid sequence of aS
consists of 140 residues with 7 copies of an unusual 11- residue repeat, followed by
hydrophobic tail (Fig.2).
3

Figure 1: Model of α-synuclein aggregation and Lewy body formation. Figure taken from
(24).

This 11- residue repeat of the protein is highly conserved whereas the carboxyl
terminal domain consisting of acidic residues is less-conserved. The 11- residue repeat at
the amino terminal make up a apolipoprotein-like class-A
2
helix which mediates binding
to phospholipid vesicles; lipid binding is accompanied by a large shift in protein
secondary structure, from around 3% over 70% α-helix (33).
When aS was found to be bound to the membranes it adopted an α-helical
confirmation (15). In solution monomeric aS was found to be highly dynamic and was
classified as natively unfolded (44).
The structure of aS has been studied by various techniques and has been shown
that the cores of aS fibrils are formed by residues ~34 to ~101, the N- and C-terminal
domains remain distorted (11, 12, 20).
4

Figure 2: Amino acid sequence of α-synuclein with the repeat sequences illustrated in
Roman numerals.

It has also been showed that aS forms a secondary structure which is mostly of
helical nature upon association with negative charged detergent micelle surfaces in which
the repeat regions medicates the interaction while the hydrophobic tail region remains
free in the solution (41).
The structural and morphological characteristic features of these filaments are
very similar to the aS filaments in the human brain (7, 14). The assembly of recombinant
aS is dependent on the nucleation (6, 13). The assembly is also hierarchical, occurring
from the first 100 residues of aS (12, 29). In contrast the carboxy-terminal of aS is said to
inhibit the fibril formation (7, 30, and 38). In amyloid fibrils aS adopts a similar β-strand
confirmation to that of the Lewy body when they are found in aggregated form (12). A
lipid environment promotes aS to form dimers and multimers that are in α-helical form
(5). The A53T increases the rate of assembly of the aS fibrils, which indicates that it is
the primary reason for the fibril formation (32). The A30P mutation has varied effects on
5

fibril formation. Varied results of increase, no change and inhibitory effects are observed
in this case (6, 38).

1.4 Function of aS:
Majority of the aS molecule is found to be bound to small unilammelar vesicles
(SUV) of size 300-400 Å which are structurally very similar to the synaptic vesicles (8).
aS has a very major role in synaptic plasticity and in the release of neurotransmitter (23).
It is also found that aS can act as a molecular chaperone complementing the chaperone
activity of CSPα (3). This suggests that aS helps in maintaining the overall integration of
the synapse. CSPα is important for integrity of the synapse and for maintaining normal
levels of SNARE complexes. SNARE complexes are important in release of
neurotransmitters into the synaptic cleft which is followed by the creation of action
potential (21, 43). CSPα is also found to play a key role in folding of SNARE complexes
(3). The up regulation of aS is compensated for loss of CSPα activity by which the
degeneration of pre-synaptic vesicles is prevented (3). This activity is exhibited by both
wild type aS and A53T mutated aS and not by A30P mutants (3). These results support
the reduced ability of the A30P mutant to interact with lipid membranes (41).

6

2. BACKGROUND AND AIM OF THE STUDY:
Solution NMR (Nuclear Magnetic Resonance) technique along with TROSY
(Transverse Relaxation Optimized Spectroscopy) can be used in numerous studies to
tackle the fundamental problem of large biomolecular proteins and their structure
determination (36). When a protein molecule interacts with small molecules there are
considerable changes in the chemical environment of their nuclei. These small changes
act as a specific change for the NMR to locate the binding of the protein.
The aS family of proteins bind to synaptic vesicles using an amphiphilic helix (7,
22). aS is usually unstructured in absence of a membrane and lipids but these proteins
become structured when they associate with lipids or membranes (8). Because of the
large size it is not possible to find the structure of aS interacting with vesicles using
solution NMR, detergent micelles can be used and they are very good mimics of the
membranes (41, 42).
In presence of the detergent sodium lauroyl sarcosine (SLS), (Fig.4), (37), a good
spectra is observed both below and above the Critical Micelle Concentration (CMC)
which proves a stable protein-detergent interaction.

7

Figure 3:Structure of SDS-micelle bound α-synuclein (42).

Previous studies of Dr.Ulmer have shown that aS binds to SDS- micelle and NMR
studies showed that the repeat region of aS consisted of two curved α-helices that are
anti-parallel in nature, connected by a short extended region and an unstructured mobile
tail in the C-terminus (Fig 3), (42).

Figure 4: Chemical structure of SLS- sodium lauroyl sarcosine.
IUPAC name: N-methyl-N (1-oxododecyl)-glycine, sodium salt.

It is also shown that the detergent acts as a support for the folding of aS (41, 42).
Furthermore dynamics of the bound aS to the SDS-micelle is found to be increased in the
regions if glycine residues of aS (Fig. 2), (41, 42). The various lysine side chain residues
of aS are found to be involved in electrostatic interactions with the anionic lipid groups of
the SUVs and micelle (12, 41).
8

The backbone dynamics vary distinctly within the helical regions of micelle
bound aS (42). Differences in structure and dynamics of the helices of aS are of great
importance in the function of aS. Residues in the region Ala
30
-Val
37
and the connector of
the aS helices shows a considerable amount of backbone dynamics (42). It is also
identified that three regions Ala
30
-Ser
42
, Asn
65
-Val
70
, and Glu
83
-Ala
89
are of reduced
backbone order (42). Common to all three regions are Gly residues in close proximity,
namely Gly
31, 36, 41
, Gly
67, 68
, Gly
84, 88
.
In consideration to the above fact, this study specifically aims to mutate the Gly
residues in the first two regions, Gly
31, 36, 41
and Gly
67, 68
. These glycine (Gly) residues are
mutated to alanine (Ala) and a suitable detergent system using SLS is created for solution
NMR spectroscopy. Comparison of structure and dynamics of the mutated aS variants to
aS wild type provides an insight to the role of these Gly residues in the helix formation of
the micelle-bound aS.

9

3. MATERIALS AND METHODS:
3.1 Production of aS variant constructs:
The human aS gene was expressed in ampicillin restricted, Tlac promoter
controlled pET-41 vector (Novagen, Inc.). The aS mutant variants are prepared using
QuickChange Mutagenesis (Stratagene, Inc.). Quickchange mutagenesis technique is
used to create the variants of aS with three point mutations in the positions 31, 36 and 41.
These glycine residues are changed into alanine and this variant is named as aSG(III).
The second mutant is named as aSG(II) which has two point mutations at glycine 67, 68
mutated to alanine. QuickChange point mutation is done using Pfu-Turbo DNA
Polymerase (Stratagene, Inc.) using the PCR Thermal Cycler (Bio-Rad i-Cycler,
Hercules, CA). The Pfu-Turbo DNA Polymerase replicates both the high fidelity
plasmids without displacing the mutant oligonucleotide primers (IDT, Coralville) that are
designed to create the point mutation. In this process the double stranded DNA vector
utilizes the two oligonucleotide primers to make the point mutation. These
oligonucleotide primers (IDT, Coralville) are designed complimentary to the vector with
the change in the position of mutation and is extended using the Pfu-Turbo DNA
polymerase. After the extension using the Pfu-Turbo DNA Polymerase the constructs are
cut using Dpn-I endonuclease (New England Biosciences), specific for methylated and
hemi-methylated DNA sequences which digests the parental DNA sequence. This DNA
is then extracted from the cells using PCR Purification Kit (Qiagen, Inc.). The isolated
DNA is then transformed into XL-10 ultra-competent cells (Stratagene, Inc.). The DNA
from the transformed cells are then isolated using DNA extraction kit (Qiagen, Inc.). The
10

extracted DNA is then given for sequencing at the DNA core facility at Norris Cancer
Institute of USC. Similar procedure is followed for the other variant too. Both the
variants with point mutations are named as aSG(III) and aSG(II).

3.2 Expression of the aS variants:
3.2.1 Expression of unlabelled aS variants in Luria-Bertani (LB) media:
The mutants aSG(III) and aSG(II) were over expressed in Escherichia coli
BL-21(DE3) Plys-T1R cells and were cultured in Luria-Bertani, LB-broth Lennox
((EMD, Germany), 20 g/liter). The culture media were prepared using Milli-Q water
(Filtering unit Milli-Q Biocel with Filter Millipak Express 20, 0.22μm, Millipore,
Eschborn) and autoclaved for sterilization (20min, 1.2 bar, 120
o
C). A selective media is
prepared by adding sterile-filtered (0.2 μm) ampicillin of final concentration 100μg/ml to
the media when the temperature is approximately 50
o
C. The aS variants are first
transformed and then sub-cultured in 2mL of Luria-Bertani media for 8hrs at 37
o
C at 180
rpm. After growing it for 8hrs 500μl of the culture is transformed to 50 mL starter culture
and is grown over night at 37
o
C, 180 rpm. 25mL of the overnight culture is spun down
for 5 mins, 2500xg at room temperature and resuspended with fresh LB and transformed
into 1L culture, it is also made sure that the culture flask have enough head space. A 4L
culture flask is used. During the growth hours the optical density (OD
600
) of the culture
media is constantly monitored. In these conditions, the OD
600
reaches 1 in 3 hrs and its
now the culture is induced with 1mL of IM IPTG (Isopropyl-β-D-thiogalactopyranoside)
11

is dissolved in Milli-Q water (2.38g/10mL) to a final concentration of 1M and is grown
for 3hrs at 37
o
C at 180 rpm.
3.2.2 Expression of
15
N labeled aS variants in M9-Minimal medium:
Both the mutants aSG(III) and aSG(II) were over expressed in E.coli BL21(DE3)
cells and were cultured in M9-Minimal media. The composition of the M9-Minimal
media is as follows; a 20X stock of 120g Na
2
HPO
4
. 7H
2
O, 60g KH
2
PO
4
, 10g NaCl is
made up to 1L. 1L M9-Minimal medium is done by adding 50mL of the 20X stock, 1mL
of 1M MgSO4 for a final concentration of 1mM in 1L of the minimal medium. 1mL of
0.1M CaCl
2
for a final concentration of 100μM final concentration in 1L of the minimal
medium is added. 0.5g of isotope labeled NH
4
Cl
2
salt (Cambridge Isotope Laboratories,
UK) is added. All the stock solutions are prepared using Milli-Q water (Filtering unit
Milli-Q Biocel with Filter Millipak Express 20, 0.22μm, Millipore, Eschborn). The media
is then made up to 1L using Milli-Q water. It is then autoclaved for sterilization (20min,
1.2 bar, 120
o
C). The media is made selective by adding sterile-filtered (0.2 μm)
ampicillin of final concentration 100μg/ml to the media when the temperature is
approximately 50
o
C and 10 mL of 20% sterile glucose solution is added. 1mL of thymine
from 10mg/mL stock (100mg of Thymine is dissolved in10 mL of sterile Milli-Q water)
is added to 1L of the minimal media. The aS variants are first transformed and then sub
cultured in 2mL of Luria-Bertani media for 8 hrs at 37
o
C at 180 rpm. After growing it for
8 hrs 500μl of the culture is transformed to 50 mL starter culture (Minimal Media) and is
grown over night at 36
o
C, 180 rpm. 25mL of the overnight culture is spun down for 5
mins, 2500xg at room temperature and resuspended with fresh Minimal Media and
12

transformed into 1L culture, it is also made sure that the culture flask has enough head
space. A 4L culture flask is used. During the growth hours the optical density (OD
600
) of
the culture media is constantly monitored. When the OD
600
of the culture reaches 1, the
culture is induced with 1mL of 1M IPTG (Isopropyl-β-D-thiogalactopyranoside) and is
grown for 3hrs at 37
o
C at 180 rpm.

3.2.3 Expression of
13
C,
15
N labeled aS variants in M9-Minimal media with D
2
O:
In prior to the making the M9-Minimal media with D
2
O salt exchange is performed
for the making of the media. 3.2g of Na
2
HPO
4
, 3g of KH
2
PO
4
, 0.5g of NaCl, 0.5g of
15
N
(isotope labeled) NH
4
Cl
2
(Cambridge Isotope laboratories, UK), 0.2467g of
MgSO
4
.7H
2
0, 0.0147g of CaCl
2
.2H
2
0, 0.01g of Thymine chloride and 3g of
13
C-isotope
labeled glucose (Cambridge Isotope Laboratories, UK) is added and are well mixed with
25 ml of D
2
O. The mixed salts are then subjected to heat applied dissolving at 70
o
C for
few minutes until the salt completely dissolve in D
2
O. The dissolved salts are then frozen
using liquid nitrogen and are then freeze-dried using a freeze-drier to make the mixture
completely anhydrous. Once the mixture is completely freeze-dried they are added to 1L
of D
2
O in a 4L flask. It is made sure that the whole process is done in sterile environment
void of moisture or water particles. The media is made selective by adding sterile-filtered
(0.2 μm) ampicillin (made with D
2
O) of final concentration 100μg/ml to it. The aS
variants are first transformed and then sub-cultured in 2mL of Luria-Bertani media for 8
hrs at 37
o
C at 220 rpm. After its grown for 8 hrs 500μl of the culture is transformed to 50
mL starter culture (M9-Minimal media) and is grown over night at 36
o
C, 180 rpm. 25mL
13

of the overnight culture is spun down for 5 mins, 2500xg at room temperature and
resuspended with fresh Minimal media made with D
2
O and transformed into 1L culture;
it is also made sure that the culture flask has enough head space. A 4L culture flask with
baffles is used. During the growth hours the optical density (OD
600
) of the culture media
is constantly monitored. When the OD
600
of the culture reaches 1, the culture is induced
with 1mL of 1M IPTG (Isopropyl-B-D-thiogalactopyranoside is dissolved in D
2
O
(2.38g/10mL) to a final concentration of 1m) and is grown for 3hrs at 37
o
C at 180 rpm.

3.3 Cell lysis:
The cells from the media are then harvested by centrifuging them at 4
o
C and
4000xg for 20 mins (SS-6000 rotor, Sorvall). The supernatant is removed and the cell
pellets are resuspended in 12 mL of 50mM Tris HCl pH 7.5, 500mM NaCl per 1L of the
culture. It is resuspended, solution with the cell pellets are homogenous without any
suspended cell particles. Using 15mL tubes the lysed culture is heat precipitated at 80
o
C
for 14 mins, interrupted by constant inverting of tubes at an interval of 2 mins to keep the
cell well suspended. Then the lysed mixture is cooled on ice for 15 mins. After cooling
the samples are centrifuged at 16,000 rpm (SS-34 Rotor, Sorvall) for 30 mins at 4
o
C. The
supernatant is then sonicated for a minute to shear the genomic DNA.

14

3.4 Purification and Characterization of aS:
3.4.1 Cation exchange chromatography:
The recombinant over expressed variants of aS was purified using cation
exchange chromatography. The AΚTA
®
purifier Prime plus FPLC (Fast Performance
Liquid Chromatography) system (Amersham Biosciences, Freiburg) is employed for the
purification. A HiTrap
®
Q-Sepharose (GE Healthcare, USA) is used. HiTrap
®

Q-Sepharose columns consist of high performance beads of sepharose which are
negatively charged due to the presence of sulfate groups. Before the start of the
purification the AΚTA
®
system is washed using system wash option to make sure that
the system is void of unnecessary buffers or solutions. After the system wash, the column
is attached to the system. A 5-column volume (CV, 1-CV is 5mL) run is performed using
Milli-Q water. This wash is followed by equilibration of the column using 50mM Tris pH
7.5, 50mM NaCl at a flow rate of 5mL/min. The column is equilibrated until a stable base
line is reached in the chromatogram (Prime plus Viewer, GE Life Sciences, USA). The
supernatant which is sonicated is diluted 1:10 with 50mM Tris HCl pH 7.5. The diluted
solution is now loaded onto the column at a flow rate of 4mL/min. After loading the
diluted supernatant the column is washed with 6-coumn volumes (CV) of 50mM Tris
HCl pH 7.5, 50mM NaCl. It is then washed with 6-CV of 50mM Tris HCl pH 7.5,
100mM NaCl followed by another 6-CV wash of 50mM Tris HCl pH 7.5, 150mM NaCl
to remove unbound proteins..
The bound proteins are eluted using 50mM Tris HCl pH 7.5, 300mM NaCl. The
eluted protein solution is collected in one tube. The various fractions collected during the
15

elution are analyzed by SDS-PAGE Gel electrophoresis. After elution the column is
washed with 50mM Tris HCl pH 7.5, 1M NaCl and with Milli-Q water followed by 20%
ethanol for storage. The HiTrap Q-Sepharose column is then stored under 4
o
C.

3.4.2 Concentration of the aS protein:
The protein obtained from the cation exchange chromatography is then subjected
to concentration. Concentration of the protein is performed using a spin filter
concentrator with a membrane cut-off of 5kDa MWCO (Sartorius Vivaspin, USA). The
protein is loaded to the spin filter tube and subjected to spinning using a spin bucket rotor
centrifuge (Sorvall, Legend Mach-1.6R). It is made sure that the tubes are balanced and
the rotor is not spun for more than 4000xg. The protein is concentrated by this method to
up to 2mL. The concentrated protein is centrifuged for 10 min at max speed (13,000 rpm,
Hettich Zentrifuge, Germany, Mickro 200R) to pellet out particulate matter. The
centrifuged protein is then filtered using a 0.22 μm filter (Nalgene, USA).

3.4.3 Size exclusion chromatography:
Hi-Prep
®
26/60 Sephacryl-S100 gel filtration column (GE Life science, USA) is
used for the size exclusion chromatography to obtain pure aS. The AΚTA
®
purifier Prime
plus FPLC (Fast Performance Liquid Chromatography) system (Amersham Biosciences,
Freiburg) is employed for the purification. The gel filtration column is equilibrated
overnight at a flow rate of 0.4 mL/min using 50mM Tris pH 7.5, 300mM NaCl and
0.02% NaN
3
. All the buffers used for the Gel filtration column are filtered using 0.22μm
16

filters. It is also made sure that the injection loops (Injection loop volume- 5mL) of the
FPLC system is washed with the same above mentioned equilibrating buffer. The
concentrated filtered aS protein from the cation exchange chromatography is then loaded
on to the injection loop when the injection valve is in “Load” position. After complete
loading of the sample to the loop, the injection valve is switched over to “Inject” position
to inject the sample into the column. After loading the sample onto the column the flow
rate is increased to 1.3mL/min. The synuclein peak is then collected at an approximate of
~110 ml. After the collection of the peak proteins the column is stored in 0.22μm filtered
Milli-Q water with 0.02% NaN
3
.

3.5 Buffer exchange of aS proteins:
The collected aS proteins from gel filtration columns are in Tris pH 7.5, 300mM
NaCl and 0.02% NaN
3
buffer. For the preparation of NMR sample the aS protein had to
be exchanged with 20mM NaP pH 7.4, 0.02% NaN
3
NMR buffer. This buffer exchange
was performed by four concentration-dilution cycles using spin filter (5kDA MWCO,
Sartorius, USA) in a spin bucket centrifuge. The proteins are concentrated down to an
approximate of ~500μL.

17

3.6 Determination of protein concentration:
Protein concentrations were determined according to Beer-Lambert’s Law.
A=ε.c.d, were A is the absorption, ε is the molar extinction coefficient (M
-1
cm
-1
), c is the
concentration of the measured sample of protein (M) and d is the thickness of the cuvette
used to measure the absorption.
For the determination of the concentration of the protein the absorption was
measured at a wave length of 280 nm (A
280
). The absorption was measured using
spectrometer (Agilent Technologies, USA). Measurements were made in a black walled
quartz cuvette (Agilent Technologies, USA) with a thickness of 1cm, as a reference blank
solution the 20mM NaP pH 7.4, 0.02% NaN
3
NMR buffer is used. The molar extinction
coefficient ε used for the determination of the concentration is ε
280
= 5120 M
-1
cm
-1
.

3.7 SDS-PAGE electrophoresis:
Results of the expression of recombinant aS were analyzed by SDS-Page for their
quality and purity. The SDS-PAGE gels was casted using the in-house protocol in gel
casting chambers. The separation gel consisted of 16.5% (w/v) acrylamide, 2% (w/v)
N,N-methylene-bisacrylamide, 0.1% (w/v) SDS, 1M Tris-HCl pH 8.45. The stacking gel
consisted of 10% (w/v) acrylamide, 0.31% (w/v) N, N-methylene-bisacrylamide, 0.1%
(w/v) SDS, 1M Tris-HCl pH 8.45. The polymerization was initiated by addition of 10μL
TEMED and 100μL freshly prepared ammonium persulfate (APS) solution per 20mL of
the gel solution. The anode buffer used for the electrophoresis consisted of 0.2M Tris-
18

HCl pH 8.9 and cathode buffer with 0.1M Tris-HCl pH 8.25, 0.1M Tricine and 0.1%
w/v) SDS.
The SDS electrophoresis chamber (Bio-Rad, Hercules) was used for the
electrophoresis, and was performed under a constant voltage of 40mA. A protein marker
standard was also run along (Lonza Prosieve marker, Fischer Scientific). Samples of the
proteins to analyze are prepared by adding 1:1 with the gel loading buffer. The samples
eluted from the ion exchange chromatography and gel filtration chromatography is used.
The concentrated samples are not used. 30μL mixture of gel loading dye and sample is
loaded on to each well along with the standard and they are electrophoresed at constant
voltage of 40mA as mentioned above. The samples are completely run to the bottom and
it’s made sure the sample does not run out of the gel.
For visualization of the separated proteins the gels were incubated in a microwave
oven with protein staining solution Coomassie Brilliant Blue R-250 0.05% (w/v), 45%
methanol and 9.2% acetic acid for 2 mins. The stained gels are then destained using
destaining solution of 80% ethanol and 20% acetic acid. The destained gels were
documented using gel-documentation system (Gel-Doc 2000, Bio-Rad, Hercules, CA).

3.8 NMR spectroscopy:
3.8.1 NMR sample preparation:
The expressed, purified recombinant aS protein variants were completely buffer
exchanged and measured for concentration as mentioned in the previous sections. NMR
samples for both the proteins aSG(II) and aSG(III) are prepared with a final 0.75mM
19

protein concentration, 75mM concentration of SLS detergent (1:100 Molar ratio with the
protein) and 6% deuterium oxide (D
2
O) and were made up to 280μL using the NMR
Buffer. D
2
O is used for the field frequency lock and NaN
3
in the NMR Buffer acts as an
antimicrobial agent in the sample. Variable restricted straight NMR sample tubes
(Shigemi Company) were used. For all the NMR analysis uniformly labeled
13
C-
15
N
samples were used at a constant sample concentration of 0.75mM.

3.8.2 NMR spectrometers and measurement:
NMR measures the signal of nuclear spins in large homogenous magnetic field.
The signal is the response of spins to an applied sequence of radio-frequency separated
by a varied pulse sequence with proper time intervals. The measured signal is the sum of
the radio frequencies that have been emitted by the nuclei, which is magnetically active.
The signal decays exponentially with a characteristic time constant called the transverse
relaxation time. Analysis can be made by transforming this signal into Fourier transform
spectra which contains resonance lines that represent the various emitted radio-
frequencies. The transverse relaxation time is inversely proportional to the thickness or
the width of the emitted frequency lines which is indeed related to the size of the
molecule that is analyzed. Larger the molecular mass shorter the transverse relaxation
time and broader the lines in the resonance spectra. TROSY- Transverse relaxation-
optimized spectroscopy, improves the measurement of residual dipolar couplings and the
detection of scalar couplings across the hydrogen bonds (17). These techniques enhance
the determination of solution based structures (17).
20

All NMR experiments were recorded in Zilkha Neurogenetic institute-based
cryo-probe equipped, shielded z-gradient, five-channel Bruker-Avance
®
700-MHz
spectrometer. At 700MHz a good TROSY effect without any excessive rapid carbonyl
relaxation is obtained. TROSY improves the measurement of residual dipolar couplings
and the detection of scalar couplings across the hydrogen bonds (17) and enhance the
spectral quality.
All the NMR spectroscopic experiments and recording of NMR based data were
done by my research investigator and mentor, Tobias S. Ulmer, Ph.D.

3.8.3 NMR data processing and analysis:
Assignments of the proteins are done with the H
N
, N, C
α
, C
β
,C` spins. HNCA and
HNCACB which are optimized for CB provide the C
α
, C
β
, shifts for the residues i and
i-1. All the assignments are were done using the CARA
®
software. Pulse programs used
for the NMR experiments were optimized for the respective spectrometer based on the
fundamental experiments done by Dr. Tobias S. Ulmer, Zilkha Neurogentic Institute,
USC.

3.8.4 Secondary structure determination by NMR spectroscopy:
The method employed to find the secondary structure of the aS variants is based
on the study of the chemical shift values. It has been known and been proved that the
chemical shift values are directly correlated with the secondary structure of the protein
under experimentation (45). It has been shown that C
α
and C
β
, resonate downfield when
21

located in an α-helix and resonate up field when located in a β-sheet, whereas C
β and H
A

behave contrarily. The random coil chemical shift values have been described by Wishart
and Sykes (45). It is suggested by them that that the chemical shift values and the
secondary structure can be correlated by defining an accurate value called the chemical
shift index, CSI. The values of the secondary chemical shift are classified either +1, when
the chemical shift value is greater than the random coil value of the corresponding atom
or described with a -1 value when the chemical shift value observed is less. The
consensus CSI summarizes the results of the independent approach form the nuclei of
different atoms. α-helices assume per definition the value -1, whereby β-strands are
described by +1. The existence of an α-helices is suggested when a dense grouping of at
least four -1 factors is not interrupted by a positive factor of +1. On the other hand for the
formation of β-strands three consecutive positive factors of +1s are worth enough.
3.8.5 Distance restraint analysis using NMR spectroscopy
1
H-
15
N heteronuclear
NOE:
The Nuclear Overhauser Effect (NOE) arises from the cross relaxation between
dipolar coupled spins as a result of spin-spin interaction through the surrounding electron
space (16). Of special importance in this respect are proton-proton distances. The back
bone motions of the proteins on the picoseconds to nanosecond timescale can be
characterized using the
1
H-
15
N heteronuclear NOE (16). A presaturation delay is applied
on the amide protons, during which dipolar interactions occur between the saturated
amide protons and their bound nitrogen. It follows a
1
H-
15
N HSQC where the intensity of
the NH peaks is directly correlated to the protein flexibility.
22

1
H-
15
N heteronuclear NOE values were determined using the pulse sequence of
Dayie and Wagner with the relaxation delay of 6s including the 3s saturation period with
120
o
high powered pulses for the saturated sub spectrum (10).
1
H-
15
N heteronuclear NOE
were obtained from the ratios of peak intensities in the standard saturated spectrum to
those in the unsaturated spectrum. Peak intensities were measures and analyzed using the
peak picking routine built into NMR View software™.

23

4. RESULTS:
4.1 Construction of the aS mutants, aSG(III) and aSG(II):
From the previous works of Dr.Ulmer, et.al., (41) it was found that the region
around the III repeat of aS were more dynamic in the micelle bound structure of aS.
Apart from these regions there was also an observable change in dynamics in other
regions around the tail near the VI repeat of aS. To identify the contributions of the
Glycine (Gly) residues found in these regions, the Gly residues were mutated to Alanine
(Ala). These observations led to the construction of two mutants (Fig. 5). The aS mutant
variants were prepared using QuickChange Mutagenesis (Stratagene, Inc.). Quickchange
point mutation technique was used to create the variants of aS with three point mutations
in 31, 36 and 41. These glycine residues are changed into alanine and this variant is
named as aSG(III). The second mutant is named as aSG(II) which has two point
mutations; glycine 67, 68 mutated to alanine.

4.2 Expression and purification of recombinant aS variants:
The human aS gene was expressed in ampicillin restricted, Tlac promoter
controlled pET-41 vector (Novagen, Inc.). The mutants aSG(III) and aSG(II) were over
expressed in Escherichia coli BL21(DE3) Plys-T1R cells and were cultured in Luria-
Bertani, LB broth Lennox ((EMD, Germany), 20 g/liter). Expression in LB broth Lennox
was performed to check the quality and quantity of expression. The mutants were
expressed again in M9-Minimal media and in M9-Minimal media with D
2
0. As a result
24

of the latter a uniformly labeled
13
C,
15
N,
2
H samples of aSG(II) and aSG(III) were
obtained.

4.3 Cation exchange chromatography:
The lysed cell cultures were subjected to cation exchange chromatography. The
column is equilibrated until a stable base line is reached (Fig. 6) and then the sonicated
diluted sample termed the “Load” is allowed to enter through the column for the selective
strong binding of the aS molecules with the resin. The required aS protein eluted with
50mM Tris HCl pH 7.5, 300mM NaCl is represented by the sharp lead peak at the 150mL
(Fig. 6). The chromatogram (Fig. 6) is the cation ion exchange chromatography using the
Q-Sepharose column for the aSG(III) culture in M9 Media with D
2
O the rest
chromatograms for the other mutant and for the M9 Minimal media is not represented as
it follows the very same similar pattern.

25

Figure 5 : The sequences of the aS(wt) and the two mutant variants aSG(III) and aSG(II).
The Gly residues to be mutated are highlighted in yellow for aSG(III) and in blue for
aSG(II). The mutated residues into Ala are also highlighted in the same fashion.

26

Figure 6: Chromatogram of the Cation Ion Exchange Chromatography. The flat region
marked ‘A’ represents the stable base line as the result of equilibration. The increase in
the absorbance caused due to the loading of sample on to the columns is represented by
‘B’ followed by region ‘C’ representing the wash. The peaks ‘D’ and ‘E’ are because of
the elution with 50mM Tris HCl pH 7.5, 100mM NaCl and 50mM Tris HCl pH 7.5,
150mM NaCl respectively. The final peak marked with ‘F’ and ‘G’ represents the elution
of the aSG(III) with 50mM Tris HCl pH 7.5, 300mM NaCl.
27

Figure 7: Chromatogram of Size exclusion chromatography using Sephacryl-S100 gel
filtration column. The region marked ‘H’ in the chromatogram represents unwanted
impurities or degradation products which are not our protein of interest. The peak marked
with ‘I’ and ‘J’ from the volume ~110 mL to 125mL is the aSG(III) peak of our interest
with aS proteins.

28

4.4 Size exclusion chromatography:
Hi-Prep
®
Sephacryl-S100 gel filtration column (GE Life science, USA) is used
for the size exclusion chromatography to obtain pure aS. The overnight equilibrated
column is loaded with the concentrated and filtered protein sample obtained from the
Cation exchange chromatography. The aS peak is obtained when the total flow volume
had reached at an approximate of ~110mL (Fig. 7). The chromatogram (Fig. 7) explained
here for the for the aSG(III) culture in M9 Media with D
2
O the rest chromatograms for
the other mutant and for the M9 Minimal media is not represented as it follows the very
same similar pattern.
The protein sample eluted using both the Q-Sepharose column-cation exchange
chromatography and Sephacryl-S100 gel filtration column is analyzed using SDS-PAGE
gel electrophoresis (Fig. 8). The lane which says ‘Marker’ has the Prosieve protein
marker which is run as the standard.

29

Figure 8 : The lane-1: elution with 50mM Tris HCl pH 7.5, 100mM NaCl; represented as
“D” in the chromatogram (Fig.6), the lane-2: wash with 50mM Tris HCl pH 7.5, 50mM
NaCl and it is represented as “ C” in the chromatogram (Fig.6), the lane-3: elution with
50mM Tris HCl pH 7.5, 150mM NaCl; represented as “E” in the chromatogram (Fig.6),
the lanes 4 and 5: are the elution peaks from the size exclusion chromatography using the
Sephacryl-S100 gel filtration column; represented as “I” and “J” in the chromatogram
(Fig.7). Elution from Q-Sepharose column with 50mM Tris HCl pH 7.5, 300mM NaCl;
represented as “F” and “J” in the chromatogram (Fig.6).

30

4.5
1
H-
15
N TROSY of aSG(II) and aSG(III):

1
H-
15
N TROSY - Transverse relaxation-optimized spectroscopy, of aSG(II) and
aSG(III) were recorded. TROSY enhances the dipolar coupling of the hydrogen bonds.
The dispersed spectral map of TROSY suggests that both the mutants aSG(III) and
aSG(II) are well structured. Clear distinctive peaks of both the spectra confirms that the
both the mutants are well bound to the detergent, SLS. Even though SLS molecule
represent the large fraction, isotope labeling of the aS variants made it possible to detect
NMR signals with less interference from SLS. Thus it is made sure that the spectrum
obtained is as a result of the interaction of the mutant aS variants with the SLS micelles.
By overlaying the TROSY spectra of aSG(III) (Fig. 9) and aSG(II) (Fig. 10) with
the aS(wt) (TROSY spectral and analysis data were provided by Dr.Nageshwara Rao
Jampani, Research Associate in Dr.Ulmer’s lab, Zilkha Neurogenetic Institute, USC) it
was observed that there was a considerable shift in the peaks of the mutants with the wild
type. Both the variants aSG(III) and aSG(II) shows a significant variation in spectral peak
formation from the aS(wt) spectra. Detectable shift was observed in the regions around
the mutated residues. Apart, variations are observed in distant residues like Ala
56
, Gln
62
,
Ala
29
of aSG(III) (Fig. 9) and Val
55
, Thr
22
, Val
52
of aSG(II) (Fig. 10). It is very
significant from the TROSY of aSG(III) (Fig. 9) that there is a change in chemical shifts
apart from the mutated regions which shows that the mutation has an effect on the
overall structure and not localized to the regions of mutation as observed with
31

aSG(II).These changes in chemical shifts also suggests that there is a change in the
chemical environment.
4.6 Normalized chemical shift changes:
Normalized chemical shift changes are expressed as weighted geometric average
of
1
HN and
15
N chemical shift changes for each residue. It’s expressed as
Δδ
norm
= (Δδ
1Η
)
2
+ 0.1(Δδ
15N
)
2

Normalized chemical shift changes larger than 0.04ppm are considered significant (19). It
is observed that apart from the strong chemical shift changes of the mutated residues,
Val
38
and Ala
69
shows the maximum change of ~3.5 and ~3 for aSG(III) and aSG(II)
respectively (Fig. 11 & 12). These changes of aSG(III) (Fig. 11) suggest that the change
in shifts are observed over both the helices. Whereas in case if aSG(II) (Fig. 12) it is seen
only in the localized areas of mutation.

32

Figure 9:
1
H-
15
N TROSY overlay of aSG(III) in presence of SLS with the aSG(wt) with
SLS. The spectra of aSG(wt) is shown in black while the spectra of the aSG(III) is shown
in red color. Both the spectra were recorded with protein concentration of 0.75mM with
75mM SLS concentration. All the data were recorded using
1
H/
13
C/
15
N labeled aSG(wt)
and aSG(III) in 20mM NaP buffer, pH 7.4, 0.02% NaN
3
solution at 25
o
C and a
1
H
frequency of 700MHz. Each peak on the spectra represents an amino-acid in the protein.
33

Figure 10:
1
H-
15
N TROSY overlay of aSG(II) in presence of SLS with the aSG(wt) with
SLS. The spectra of aSG(wt) is shown in black while the spectra of the aSG(II) is shown
in red color. Both the spectra were recorded with protein concentration of 0.75mM with
75mM SLS concentration. All the data were recorded using
1
H/
13
C/
15
N labeled aSG(wt)
and aSG(II) in 20mM NaP buffer, pH 7.4, 0.02% NaN
3
solution at 25
o
C and a
1
H
frequency of 700MHz. Each peak on the spectra represents an amino-acid in the protein.
34

Figure 11: Normalized weighted chemical shift changes for aSG(III). Normalized
chemical shift changes larger than 0.04ppl are considered to be significant. The chemical
shafts of the mutant residues are not shown and are indicated by “X”.
35

Figure 12: Normalized weighted chemical shift changes for aSG(II). Normalized
chemical shift changes larger than 0.04ppl are considered to be significant. The chemical
shifts of the mutant residues are not shown and are indicated by “X”.

36

4.7 C
α α α α
chemical shifts:
The C
α
chemical shifts of nuclei are very sensitive and variations in the chemical
shifts are good information explaining the variation in structure of the protein. Chemical
shift data are derived from the HNCA and HNCACB experiments in which the
1
H
N
back
bone amide proton and the secondary shift of
13
C
α
chemical shifts for each amino acid
residue is compared between the variants aSG(III), aSG(II) with the wild type aS(wt).
13
C
α
depends on the degrees of backbone freedom, angles phi (ϕ) and psi (ψ). The angle
phi (ϕ) describes rotation about the C
α -N bond of the amino acid, and the angle psi (ψ)
denotes rotation about the bond linking the C
α
and the carbonyl carbon. A detectable
13
C
α

chemical shift increase (Fig. 13) in the variant aSG(III) from the Gly
25
residue till Ser
42

residue suggests that there is a very significant increase in the helical character when
compared to the wild type, aSG(wt) which suggests that the mutation has affected the
helical propensity in both the helices of aS. Interestingly there is an observed decrease in
13
C
α
 chemical shifts in the regions between Lys
45
to Lys
60
, it is to be noted that these
residues are distant from the mutated residues. Though the decrease is not of very high
value, a significant decrease shows that there is a decrease in helical character in these
regions which may be because of the surface of the micelle. In case of aSG(II) variant, a
considerable amount of decrease in helical character is observed form the regions of
Glu
46
to Val
52
amino-acid residues (Fig 14). Whereas the decrement increases further till
the Lys
60
residue in a very significant manner. There is an increase in the helical
character in a very high rate from the Glu
61
to the Thr
75
residue localized only to the
mutations.
37

Figure 13: Comparison of secondary C
α
chemical shifts of SLS-bound aSG(wt) and
aSG(III). The secondary chemical shifts of aSG(wt) are shown in black whereas the
secondary chemical shifts of aSG(III) are shown in red color. For both the spectra
recorded the protein-micelle complex’s concentration was 0.75mM and 75mM
respectively. All the data were recorded using
1
H/
13
C/
15
N labeled aSG(wt) and aSG(III)
in 20mM NaP buffer, pH 7.4, 0.02% NaN
3
solution at 25
o
C and a
1
H frequency of
700MHz.Secondary Chemical shifts patterns explain secondary structure of the protein.
Depending on the secondary structure, the chemical shift values of amino acids are
shifted from its random coil values. If their difference ( ∂
(Str.Protein)
- ∂
(Random coil)
) values
are positive then it’s an α-helix were as negative represents the formation of β-sheets.
More the difference more the helical propensity or the vice-versa.

38

Figure 14: Comparison of secondary C
α
chemical shifts of SLS-bound aSG(wt) and
aSG(II). The secondary chemical shifts of aSG(wt) are shown in black whereas the
secondary chemical shifts of aSG(II) are shown in red color. For both the spectra
recorded the protein-micelle complex’s concentration was 0.75mM and 75mM
respectively. All the data were recorded using
1
H/
13
C/
15
N labeled aSG(wt) and aSG(II) in
20mM NaP buffer, pH 7.4, 0.02% NaN
3
solution at 25
o
C and a
1
H frequency of 700MHz.
Secondary Chemical shifts patterns explain secondary structure of the protein.
Depending on the secondary structure, the chemical shift values of amino acids are
shifted from its random coil values. If their difference ( ∂
(Str.Protein)
- ∂
(Random coil)
) values
are positive then it’s an α-helix were as negative represents the formation of β-sheets.
More the difference more the helical propensity or the vice-versa.

39

4.8
1
H-
15
N Heteronuclear NOE:
The back bone dynamics of the proteins on the picoseconds to nanosecond
timescale can be characterized using the
1
H-
15
N heteronuclear NOE (16).
1
H-
15
N Het-
NOE enables us to characterize the dynamics of the proteins. The
1
H-
15
N Het-NOE
spectra were analyzed for both the mutants aSG(III) and aSG(II). Overlay of the aSG(III)
(Fig. 15) and aSG(II) (Fig. 16) with the aS(wt) spectra shows ( the
1
H-
15
N Het-NOE
chemical shift data were provided by Dr.Nageshwara Rao Jampani, Research Associate
in Dr.Ulmer’s lab, Zilkha Neurogenetic Institute, USC). A significant change in the back
bone dynamics is observed for the variant aSG(III) (Fig. 15). There is an increase in the
1
H-
15
N Het-NOE values, which suggest the probability of rigidification of the helix with
the micelle in the regions Ala
17
to Val
37
. Decrement in rigidity is followed in the distant
regions from Val
40
to Lys
60
. The
1
H-
15
N Het-NOE of aSG(II) (Fig. 16) has an increase in
the
1
H-
15
N Het-NOE values in the regions of the mutation from Thr
54
to Ala
69
alone.
40

Figure 15: Overlay plot of
1
H-15N Het-NOEs of aSG(III) and aSG(wt) with
micelle complex. The
1
H-15N Het-NOEs of aSG(wt) is shown in black and the
1
H-15N Het-NOEs of aSG(III) are shown in red. For both the spectra recorded the
protein-micelle complex’s concentration was 0.75mM and 75mM respectively.
All the data were recorded using
1
H/
13
C/
15
N labeled aSG(wt) and aSG(III) in
20mM NaP buffer, pH 7.4, 0.02% NaN
3
solution at 25
o
C and a
1
H frequency of
700MHz. Using Het NOEs it is possible to determine the distance between two
nuclei, the overall molecular motion and internal motions in the protein.
Dynamics of the protein is studied using Het NOE spectra. Increase in
1
H-15N
Het-NOE values suggest that the structure becomes more rigid and less dynamic.

41

Figure 16: Overlay plot of
1
H-15N Het-NOEs of aSG(II) and aSG(wt) with
micelle complex. The
1
H-15N Het-NOEs of aSG(wt) is shown in black and the
1
H-15N Het-NOEs of aSG(II) are shown in red. For both the spectra recorded the
protein-micelle complex’s concentration was 0.75mM and 75mM respectively.
All the data were recorded using
1
H/
13
C/
15
N labeled aSG(wt) and aSG(II) in
20mM NaP buffer, pH 7.4, 0.02% NaN
3
solution at 25
o
C and a
1
H frequency of
700MHz. Using Het NOEs it is possible to determine the distance between two
nuclei, the overall molecular motion and internal motions in the protein.
Dynamics of the protein is studied using Het NOE spectra. Increase in
1
H-15N
Het-NOE values suggest that the structure becomes more rigid and less dynamic.

42

5. DISCUSSION:
Pre-synaptic vesicles are the physiological targets of aS which are of diameter
50nm . By using a SLS-micelle model as a mimic to the vesicle, the aS variant-SLS
micelle complex is been studied here. Though the size of the SLS-micelle is not as big as
the vesicle, it acts a good mimic because the chemical make-up of the polar head groups
of SLS-micelle complex is similar to that of the pre-synaptic vesicles. Verification of
Thr
59
-Ala
90
residues in the aS-detergent system and aS-SUV system (negatively charged,
300-400 Å in diameter) (22) proves that the structure of amino acid residues of the aS
side chains interacting with the vesicles and aS-detergent system remains constant.
Preliminary studies of micelle bound aS were carried out using SDS-micelle. SLS-
micelle system is preferred over SDS (aggregation number 70) because it provides a
bigger surface area for binding with an aggregation number of 100.
The solution NMR study results of the SLS micelle bound aS variants (aSG(II)
and aSG(II)) is similar to that of wild type with an increase in the helical configuration in
the mutated regions. Though the chemical make-up of the SLS micelle does not affect the
structure of aS, the diameter and the size of the micelle may affect the interaction of aS
with it (22). A discontinuity in the helical confirmation of micelle-bound-aS system has
been reported near Ser
42
-Thr
44
residues (2, 3). This discontinuity were attributed towards
the smaller size of the micelle system (12)

. Preliminary studies with of aS-SDS micelle-
complex show few sub-zero C
α
chemical shift deviations denoting the break in the helix.
With SLS-micelle system the C
α
chemical shift change is of a lesser gradient. Change in
43

the shape of the micelle and the interaction of aS with the micelle contributes to these
observable changes.
Binding of aS with the vesicles and micelles makes them structured. Structural
variations in the helical confirmation and dynamics are of great importance for the
function of aS. Predominant changes in the C
α
chemical shifts in regions distant from the
mutated residues are elucidated by the comparison of the normalized weighted shift
changes for aSG(III) and aSG(II) with the wild type. Further the helical configuration of
the mutants increase in these mutated regions in both the variants and decreases further
down in the distant residues. Compensation of Gly with an Ala in an α-helix increases
the helical conformation and consistently stabilizes it in the mutated regions and buries
more non-polar region upon folding because of its lesser backbone conformational
flexibility and hence, decreased entropic cost of adopting helical conformations (26).
Thus enabling better binding with the micelle surface. This is very significant from the
1
H-
15
N-NOEs (Fig 15 & 16). As the rigidity and the helical configuration of the mutants
increase, both aSG(III) and aSG(II) forms a stable rigid helix than the wild type over the
micelle making it less dynamic and flexible. This leads to occupancy of more space over
the curvature of the micelle and restricting it to strict rigid confirmation. The increased
occupancy over the micelle is compensated by a decrement in the helical confirmation
and rigidity in the distant regions of Val
40
to Ala
53
in aSG(III) and Thr
54
to Lys
60
in
aSG(II). The small size of the micelle may also be the reason for this, thus restricting aS
to configure with the micelle surface. Interestingly incase of aSG(III) the mutations has
affected both the helices of the micelle-bound structure and making it more favorable to
44

bind with the micelle. The helical propensity is also increased throughout the region and
dynamically the regions are more rigidified. Whereas in case of aSG(II) the mutations
affects the chemical shifts and dynamics only in the local region. From these results it can
be concluded that the binding affinity of aS to the micelle is in the order aSG(III) >
aS(Wild-type) > aSG(II).

45

6. CONCLUSIONS:
It is well documented that micelles and vesicles induce and promote the
aggregation of aS proteins from random coiled monomer to dimers, trimers and
multimers (4, 25, 31). The three regions Ala
30
-Ser
42
, Asn
65
-Val
70
, and Glu
83
-Ala
89
that
are of reduced backbone order (42) are the areas of prime interest. The common feature
to all three regions are Gly residues in close proximity, namely Gly
31,36,41
, Gly
67,68
,
Gly
84,88
. The mutant asG(III) with mutated Gly
31,36,41
residues binds in a better way than
the wild-type whereas asG(II) with mutated Gly
67,68
binds weaker. From the results
obtained it is significant that these Gly residues play a significant role in the stability and
dynamics of the aS bound to the micelle.

46

REFERENCES:

1. Abeliovich, A., Y. Schmitz, I. Farinas, D. Choi-Lundberg, W. H. Ho, P. E.
Castillo, N. Shinsky, J. M. Verdugo, M. Armanini, A. Ryan, M. Hynes, H.
Phillips, D. Sulzer, and A. Rosenthal. 2000. Mice lacking alpha-synuclein
display functional deficits in the nigrostriatal dopamine system. Neuron 25:239-
52.
2. Bussell, R., Jr., and D. Eliezer. 2003. A structural and functional role for 11-mer
repeats in alpha-synuclein and other exchangeable lipid binding proteins. J Mol
Biol 329:763-78.
3. Chandra, S., G. Gallardo, R. Fernandez-Chacon, O. M. Schluter, and T. C.
Sudhof. 2005. Alpha-synuclein cooperates with CSPalpha in preventing
neurodegeneration. Cell 123:383-96.
4. Cole, N. B., D. D. Murphy, T. Grider, S. Rueter, D. Brasaemle, and R. L.
Nussbaum. 2002. Lipid droplet binding and oligomerization properties of the
Parkinson's disease protein alpha-synuclein. J Biol Chem 277:6344-52.
5. Cole, N. B., D. D. Murphy, J. Lebowitz, L. Di Noto, R. L. Levine, and R. L.
Nussbaum. 2005. Metal-catalyzed oxidation of alpha-synuclein: helping to define
the relationship between oligomers, protofibrils, and filaments. J Biol Chem
280:9678-90.
6. Conway, K. A., J. D. Harper, and P. T. Lansbury. 1998. Accelerated in vitro
fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson
disease. Nat Med 4:1318-20.
7. Crowther, R. A., R. Jakes, M. G. Spillantini, and M. Goedert. 1998. Synthetic
filaments assembled from C-terminally truncated alpha-synuclein. FEBS Lett
436:309-12.
8. Davidson, W. S., A. Jonas, D. F. Clayton, and J. M. George. 1998.
Stabilization of alpha-synuclein secondary structure upon binding to synthetic
membranes. J Biol Chem 273:9443-9.
47

9. Dawson, T. M., and V. L. Dawson. 2003. Molecular pathways of
neurodegeneration in Parkinson's disease. Science 302:819-22.
10. Dayie, K. T., and G. Wagner. 1995. Carbonyl-carbon relaxation rates reveal a
dynamic heterogeneity of the polypeptide backbone in villin 14T. J Magn Reson
B 109:105-8.
11. Del Mar, C., E. A. Greenbaum, L. Mayne, S. W. Englander, and V. L.
Woods, Jr. 2005. Structure and properties of alpha-synuclein and other amyloids
determined at the amino acid level. Proc Natl Acad Sci U S A 102:15477-82.
12. Der-Sarkissian, A., C. C. Jao, J. Chen, and R. Langen. 2003. Structural
organization of alpha-synuclein fibrils studied by site-directed spin labeling. J
Biol Chem 278:37530-5.
13. Ding, T. T., S. J. Lee, J. C. Rochet, and P. T. Lansbury, Jr. 2002. Annular
alpha-synuclein protofibrils are produced when spherical protofibrils are
incubated in solution or bound to brain-derived membranes. Biochemistry
41:10209-17.
14. El-Agnaf, O. M., R. Jakes, M. D. Curran, and A. Wallace. 1998. Effects of the
mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological
properties of alpha-synuclein protein implicated in Parkinson's disease. FEBS Lett
440:67-70.
15. Eliezer, D., E. Kutluay, R. Bussell, Jr., and G. Browne. 2001. Conformational
properties of alpha-synuclein in its free and lipid-associated states. J Mol Biol
307:1061-73.
16. Farrow, N. A., R. Muhandiram, A. U. Singer, S. M. Pascal, C. M. Kay, G.
Gish, S. E. Shoelson, T. Pawson, J. D. Forman-Kay, and L. E. Kay. 1994.
Backbone dynamics of a free and phosphopeptide-complexed Src homology 2
domain studied by 15N NMR relaxation. Biochemistry 33:5984-6003.
17. Fernandez, C., and G. Wider. 2003. TROSY in NMR studies of the structure
and function of large biological macromolecules. Curr Opin Struct Biol 13:570-
80.
48

18. Galloway, P. G., P. Mulvihill, and G. Perry. 1992. Filaments of Lewy bodies
contain insoluble cytoskeletal elements. Am J Pathol 140:809-22.
19. Hajduk, P. J., J. Dinges, G. F. Miknis, M. Merlock, T. Middleton, D. J.
Kempf, D. A. Egan, K. A. Walter, T. S. Robins, S. B. Shuker, T. F. Holzman,
and S. W. Fesik. 1997. NMR-based discovery of lead inhibitors that block DNA
binding of the human papillomavirus E2 protein. J Med Chem 40:3144-50.
20. Heise, H., W. Hoyer, S. Becker, O. C. Andronesi, D. Riedel, and M. Baldus.
2005. Molecular-level secondary structure, polymorphism, and dynamics of full-
length alpha-synuclein fibrils studied by solid-state NMR. Proc Natl Acad Sci U S
A 102:15871-6.
21. Jahn, R., T. Lang, and T. C. Sudhof. 2003. Membrane fusion. Cell 112:519-33.
22. Jao, C. C., A. Der-Sarkissian, J. Chen, and R. Langen. 2004. Structure of
membrane-bound alpha-synuclein studied by site-directed spin labeling. Proc Natl
Acad Sci U S A 101:8331-6.
23. Kahle, P. J., C. Haass, H. A. Kretzschmar, and M. Neumann. 2002.
Structure/function of alpha-synuclein in health and disease: rational development
of animal models for Parkinson's and related diseases. J Neurochem 82:449-57.
24. Lashuel, H. A., and P. T. Lansbury, Jr. 2006. Are amyloid diseases caused by
protein aggregates that mimic bacterial pore-forming toxins? Q Rev Biophys
39:167-201.
25. Lee, H. J., C. Choi, and S. J. Lee. 2002. Membrane-bound alpha-synuclein has a
high aggregation propensity and the ability to seed the aggregation of the
cytosolic form. J Biol Chem 277:671-8.
26. Lopez-Llano, J., L. A. Campos, and J. Sancho. 2006. Alpha-helix stabilization
by alanine relative to glycine: roles of polar and apolar solvent exposures and of
backbone entropy. Proteins 64:769-78.
27. Manning-Bog, A. B., A. L. McCormack, M. G. Purisai, L. M. Bolin, and D.
A. Di Monte. 2003. Alpha-synuclein overexpression protects against paraquat-
induced neurodegeneration. J Neurosci 23:3095-9.
49

28. Mezey, E., A. M. Dehejia, G. Harta, N. Tresser, S. F. Suchy, R. L. Nussbaum,
M. J. Brownstein, and M. H. Polymeropoulos. 1998. Alpha synuclein is present
in Lewy bodies in sporadic Parkinson's disease. Mol Psychiatry 3:493-9.
29. Miake, H., H. Mizusawa, T. Iwatsubo, and M. Hasegawa. 2002. Biochemical
characterization of the core structure of alpha-synuclein filaments. J Biol Chem
277:19213-9.
30. Murray, I. V., B. I. Giasson, S. M. Quinn, V. Koppaka, P. H. Axelsen, H.
Ischiropoulos, J. Q. Trojanowski, and V. M. Lee. 2003. Role of alpha-
synuclein carboxy-terminus on fibril formation in vitro. Biochemistry 42:8530-
40.
31. Narayanan, V., and S. Scarlata. 2001. Membrane binding and self-association
of alpha-synucleins. Biochemistry 40:9927-34.
32. Nussbaum, R. L., and C. E. Ellis. 2003. Alzheimer's disease and Parkinson's
disease. N Engl J Med 348:1356-64.
33. Perrin, R. J., W. S. Woods, D. F. Clayton, and J. M. George. 2000. Interaction
of human alpha-Synuclein and Parkinson's disease variants with phospholipids.
Structural analysis using site-directed mutagenesis. J Biol Chem 275:34393-8.
34. Pollanen, M. S., D. W. Dickson, and C. Bergeron. 1993. Pathology and biology
of the Lewy body. J Neuropathol Exp Neurol 52:183-91.
35. Polymeropoulos, M. H., C. Lavedan, E. Leroy, S. E. Ide, A. Dehejia, A.
Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E. S. Stenroos, S.
Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W. G. Johnson,
A. M. Lazzarini, R. C. Duvoisin, G. Di Iorio, L. I. Golbe, and R. L.
Nussbaum. 1997. Mutation in the alpha-synuclein gene identified in families with
Parkinson's disease. Science 276:2045-7.
36. Post, C. B. 2003. Exchange-transferred NOE spectroscopy and bound ligand
structure determination. Curr Opin Struct Biol 13:581-8.
50

37. Sehgal, P., J. E. Mogensen, and D. E. Otzen. 2005. Using micellar mole
fractions to assess membrane protein stability in mixed micelles. Biochim
Biophys Acta 1716:59-68.
38. Serpell, L. C., J. Berriman, R. Jakes, M. Goedert, and R. A. Crowther. 2000.
Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-
beta conformation. Proc Natl Acad Sci U S A 97:4897-902.
39. Shults, C. W. 2006. Lewy bodies. Proc Natl Acad Sci U S A 103:1661-8.
40. Spillantini, M. G., M. L. Schmidt, V. M. Lee, J. Q. Trojanowski, R. Jakes,
and M. Goedert. 1997. Alpha-synuclein in Lewy bodies. Nature 388:839-40.
41. Ulmer, T. S., and A. Bax. 2005. Comparison of structure and dynamics of
micelle-bound human alpha-synuclein and Parkinson disease variants. J Biol
Chem 280:43179-87.
42. Ulmer, T. S., A. Bax, N. B. Cole, and R. L. Nussbaum. 2005. Structure and
dynamics of micelle-bound human alpha-synuclein. J Biol Chem 280:9595-603.
43. Ungar, D., and F. M. Hughson. 2003. SNARE protein structure and function.
Annu Rev Cell Dev Biol 19:493-517.
44. Weinreb, P. H., W. Zhen, A. W. Poon, K. A. Conway, and P. T. Lansbury, Jr.
1996. NACP, a protein implicated in Alzheimer's disease and learning, is natively
unfolded. Biochemistry 35:13709-15.
45. Wishart, D. S., and B. D. Sykes. 1994. The 13C chemical-shift index: a simple
method for the identification of protein secondary structure using 13C chemical-
shift data. J Biomol NMR 4:171-80.
46. Zarranz, J. J., J. Alegre, J. C. Gomez-Esteban, E. Lezcano, R. Ros, I.
Ampuero, L. Vidal, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, E.
Gomez Tortosa, T. del Ser, D. G. Munoz, and J. G. de Yebenes. 2004. The
new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body
dementia. Ann Neurol 55:164-73.

Abstract (if available)

Abstract Parkinson's disease has been associated with misfolding of the protein alpha-synuclein (aS). Previous studies of micelle bound alpha-synucleins have shown that it forms two anti-parallel helices on the micelle surface with elevated dynamics in the glycine residues of the III, V, and VI repeat of its amino acid sequence. Thus by mutating these residues to alanine a significant change in the dynamics and structure has been observed. A better micelle system has been established using sodium lauroyl sarcosine (SLS) which has more aggregation number than SDS and decreases the negative restraint on the C-alpha chemical shifts. Two aS variants, aSG(III) with mutated Gly residues at 31, 36 and 41 mutated to Ala and aSG(II) with mutated Gly residues at 67 and 68 to Ala are studied. Backbone and dynamic parameters of the variants show that there is an increase in chemical shift causing an increment in helical character in the regions of mutation. But this is been compensated at other distant residues where the same character is significantly reduced. Similar pattern is observed in the dynamic parameters which show an increase in rigidity of the helix in the mutated regions. These strong increase in chemical shift and decrease in dynamics suggest that the glycine residues in these positions play a significant role in the interaction of the protein alpha-synuclein with the lipid surfaces.

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Asset Metadata

Creator Balasubramanian, Adithya (author)

Core Title Glycine to alanine mutations affect the structure and dynamics of micelle bound alpha-synuclein

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2008-08

Publication Date 08/01/2008

Defense Date 06/25/2008

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

Tag alpha-synuclein,C-alpha,chemical shift,micelle-bound synuclein,neurodegeneration,NMR,OAI-PMH Harvest,Parkinson's disease,protein,structural biology

Language English

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

Creator Email abalasub@usc.edu,adithya85@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1507

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