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Oligomer formation of functional amyloid protein - Orb2A

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Oligomer formation of functional amyloid protein - Orb2A

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Oligomer formation of Functional
Amyloid Protein – Orb2A
By
Ninad Agashe

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2014
Copyright 2014 Ninad Agashe

ACKOWLEDGEMENTS
I would like to express my special appreciation and thanks to my
adviser Dr. Ansgar Siemer for your constant encouragement and
guidance towards research and writing this thesis. Your word of advice
on both research and my career has been very important for me. I would
also like to thank other thesis committee members Dr. Zoltan Tokes, Dr.
Ralf Langen and Dr. Ian Haworth for your guiding comments and
suggestions.
I would like to thank all member of Siemer Lab for being a
wonderful supportive team. I would like express my regards to members
of Langen Lab and Ulmer Lab for their support. I would like express
special thanks to Dr. Janet Oldak for allowing us to use Dynamic Light
Scattering facility at her lab.
Thank you everyone for your help and support.

Table of Contents
1. Introduction ………………………………………………………………..1
a. Amyloid Fibres – Background ……………………………………….1
b. Classification of Amyloids – Functional and
Pathological Amyloids ……………………………………………..2
c. Precursor Species of Amyloid Fibre …………………………............5
d. Mechanism of Amyloid Formation …………………………………..6
e. Oligomers Preceding Amyloid Fibrils ………………………..............7
f. Long Term Memory Formation ……………………………………..10
g. CPEB Protein Family is required for
Stabilizing Activity Dependent Synaptic Changes …………….........11
h. Mechanism of CPEB Dependent Long Term
Memory Formation ………………………………………………….12
i. Domain Structure of Rare Isoform Orb2A and
Predominant Isoform Orb2B ………………………………………..14
j. Central Working Hypothesis about Orb2 Amyloid Formation……...16
2. Objectives of the Project …………………………………………………18
3. Preliminary Work: Orb2A88 Validly Represents the Amyloid Core …20
4. Results …………………………………………………………………......23
a. UV Scattering Indicates Presence of Early Stage

Soluble Oligomers ………………………………………………......23
b. Orb2A88_WT and Orb2A88_F5Y Form Oligomers in
Solution before Forming Fibres ………………………….……...25
c. Early Stage Soluble Oligomers are Imaged Using
Transmission Electron Microscopy (TEM) …………………...........28
d. Developing Semi Denaturating SDS – Agarose Gel
Electrophoresis to Detect Early Stage Oligomeric Species …...........29
e. F5Y Mutation may Stabilize the Oligomeric State of Orb2A ……...34
f. Secondary Structure Analysis ………………………………….......35
5. Discussion ………………………………………………………………...37
6. Materials and Methods ……………………………………………….....44
a. DNA cloning, Transformation, Protein Expression,
Purification and Renaturation (Desalting) ……………………...44
b. UV spectroscopy ……………………………………………….......46
c. CD Spectroscopy ……………………………………………….......47
d. Electron Microscopy…………… ……………………………….…47
e. Dynamic Light Scattering (DLS) ………………………………..…47
f. SDS-Agarose Gel Electrophoresis ………………………………....49
7. References …………..................................................................................53
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Introduction
The focus of this project is to establish the formation and characterize the
early stage oligomers of the functional amyloid protein Orb2, which is critical for
long term memory formation in Drosophila. This section describes some of the
important structural and functional characteristics of amyloid fibrils and precursor
states of the mature amyloid fibrils. It also provides a classification of amyloids as
pathological and functional amyloids and the role of early stage oligomers in case
of both types of amyloids. The section describes the mechanism of long term
memory formation and role of Drosophila cytoplasmic polyadenylation element
binding protein (CPEB) – Orb2.
Amyloid Fibrils – Background
A wide range of natural and artificial peptides and proteins have an intrinsic
propensity to self-assemble into amyloid fibrillar structure highly rich in β-sheet
rich secondary structure (Knowles & Buehler, 2011). The amyloid fibrillar
structure first received attention through their association with diseases related to
protein misfolding including neurodegenerative diseases such as Alzheimer’s and
Parkinson’s disease, in which normally soluble proteins are misfolded and
deposited pathologically as aggregated structures (Chiti & Dobson, 2006; Knowles
& Buehler, 2011). The fibrils are generally organised in a very similar manner at
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molecular level; they are characterised by β-strands oriented perpendicular to the
fibril axis, and the backbone stabilized by a dense hydrogen bonding network. At
supramolecular level, the β-sheets often extend to thousands of molecular units
(Knowles & Buehler, 2011). The fibrils are imaged in vitro using transmission
electron microscopy (TEM) or atomic force microscopy (AFM). The data from
these experiments reveal that, the fibres usually consist of 2-6 protofilaments,
which are constituent units of amyloid fibrils. Each protofilament is about 2-5 nm
is diameter and are either twisted to form rope like fibrils or associate laterally in
some cases to form ribbon like structures (Chiti & Dobson, 2006; Knowles &
Buehler, 2011; Perutz, Finch, Berriman, & Lesk, 2002). High resolution structural
details can be studied using EPR, solid state NMR and X ray crystallography. The
β-sheet rich amyloid fibres have ability to bind specifically to fluorescent dye
Thioflavin T (ThT) and Congo Red (CR). This characteristic is used widely along
with EM to detect the presence of amyloid fibres (Chiti & Dobson, 2006).
Classification of Amyloids – Functional and Pathological Amyloids
A list of some of the known human diseases associated with extracellular
amyloid fibrils or intracellular inclusions with amyloid like characteristics are
given in Table 1. These diseases can be broadly classified as neurodegenerative
conditions where the aggregation occurs in Central Nervous System (CNS), non –
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neuropathic localized amyloidosis in which aggregation happens in single type of
tissue other than CNS, and non – neuropathic systemic amyloidosis in which the
aggregation occurs in multiple tissues (Bucciantini et al., 2004; Chiti & Dobson,
2006; Stefani & Dobson, 2003).
Subsequently, many functional amyloid–like materials are discovered in
varying roles throughout nature (Fowler, Koulov, Balch, & Kelly, 2007; Knowles
& Buehler, 2011). Some of the proteins in normally functioning biological system
can exist in both soluble conformation and amyloid like aggregated form. The later
state can be self-perpetuating, known as prions (Chiti & Dobson, 2006; Si,
Lindquist, & Kandel, 2003). Some of the examples of functional amyloids are also
given in Table 1. The precise molecular function of currently identified functional
amyloids remains unknown in many cases. Elucidation of regulatory mechanisms
that facilitate the use of amyloids for physiological function might give important
information for developing novel therapies for amyloid diseases (Fowler et al.,
2007).
Furthermore, a wide range of unrelated proteins and peptides can be
thermodynamically destabilized from natively folded state to artificial fibrillar
materials in vitro that are characterized by quaternary amyloid structure. This has
led to design of novel functional nanomaterial (Knowles & Buehler, 2011).
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Of the examples listed in Table 1, neuronal specific isoform of CPEB in
marine snail has been found to exist in soluble as well as amyloid aggregated form
(Si, Lindquist, et al., 2003). The amyloid aggregates are the active state, which
regulate mRNA translation and maintenance of synaptic changes and memory
storage (Heinrich & Lindquist, 2011a; Huang, Kan, Lin, & Richter, 2006; Si, Choi,
White-Grindley, Majumdar, & Kandel, 2010). The protein Orb2 also belongs to the
same family of proteins and is involved in long term memory formation in
Drosophila (Keleman, Kruttner, Alenius, & Dickson, 2007; Krüttner et al., 2012;
Majumdar et al., 2012).
Table 1. Examples of Different Forms of Amyloids
Context/Function Protein Primary localization or
Organization
Functional Amyloids
Control of transcription Ure2p Yeast
Long term maintenance of
synaptic changes
Neuron specific isoform
of CPEB
Marine snail
(Aplysia Californica)
Biofilm production Curli E. coli bacteria
Melanin biosynthesis Pmel17 Melanosomes in
mammalian skin
Pathological Amyloids
Alzheimer’s disease Amyloid β(aβ) peptide Extracellular central
nervous system (CNS)
Parkinson’s disease α-Synuclein Intracellular CNS
Type II diabetes Amylin Extracellular (pancreas)
Systemic amyloidosis Lysozyme, transthyretin,
serum amyloid A,
immunoglobulin light
chain
Various tissues including
liver, heart, spleen and
kidney

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Figure 1. Classification of amyloid materials; a. Functional amyloid in biofilm
production by some bacterial species such as E. coli or some salmonella species b.
Amyloid plaques as seen in mouse models of Alzheimer’s disease c. TEM image
of Pmel17 scaffolds in melanosomes involved in biosynthesis of melanin d. Lewy
bodies due to protein aggregation that develop in neurons in Parkinson’s disease.
Taken from (Knowles & Buehler, 2011).

Precursor Species of Amyloid Fibres
During the formation of mature amyloid fibrils, series of metastable and
transient protein state are observed. This includes unstructured aggregates and non-
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specific aggregates, prefibrillar oligomers and structured protofibrils (Chiti &
Dobson, 2006). Some of the recent findings suggest that low molecular weight
oligomers and protofibrils might be more toxic than mature fibrils (Chiti &
Dobson, 2006; Eisenberg & Jucker, 2012; Stefani & Dobson, 2003). These states
are highly dynamic in nature, metastable and transient thus it’s difficult to isolate a
homogeneous fraction, making structural and biochemical investigation of these
states difficult. The focus of this project is one of these states, the early stage
oligomers. The role of these oligomers can be understood in the context of the
mechanism of amyloid formation, toxicity of the oligomers and importance of
these oligomers for functional amyloids.
Mechanism of Amyloid Formation
Most of the available evidence indicates that the amyloid fibre formation
occurs via a nucleated growth mechanism. The time course for conversion a
protein or peptide to amyloid fibrils is determined by ThT fluorescence assay or
light scattering techniques. Such measurements show a lag phase followed by a
phase of rapid exponential growth (Chiti & Dobson, 2006). Lag phase is thought to
be the time required for nuclei formation and the fibril growth occurs via
association of monomers or oligomers with the nucleus (Chiti & Dobson, 2006;
Knowles & Buehler, 2011).
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Although fibrils do not appear during the nucleation step, the nucleation step
is thought to be very important as rate limiting step in most cases. Varieties of
oligomers forms and assemble including β-sheet rich protofibrils during the
nucleation step. These are required for nuclei and amyloid core and thus formation
of mature amyloid fibrils (Chiti & Dobson, 2006).
A variety of factors influence the length of nucleation step including
experimental conditions, mutations and presence of performed fibrils (seeding)
(Chiti & Dobson, 2006). Functional amyloids do not have a prolonged lag phase
due to their physiological role (Chiti & Dobson, 2006; Fowler et al., 2007; Stefani
& Dobson, 2003). The shortened lag phase in the context of the functional amyloid
raises possibilities about rapidly forming oligomers, which would then regulate
rapid transition to fibrillar amyloid structure.
Oligomers Preceding Amyloid Fibrils
In this section, some of the existing research regarding various amyloid
protein oligomers is reviewed. This provides a basis for the roles these oligomeric
species might play.
A lot of research has been done on the Aβ peptide owning to its association
with Alzheimer’s disease. The aggregation of this Aβ 1-42 peptide is preceded by
metastable, non fibrillar species which appear as spherical beads or beads on chain
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with diameter 2-5 nm. These protofibrils have significant β-sheet structure and can
bind to ThT and CR. These protofibrils are typically made up of ~20molecules
(Chiti & Dobson, 2006). Aβ also forms low molecular weight soluble oligomers
which are in rapid equilibrium with corresponding monomeric form (Sakono &
Zako, 2010). Aβ monomers are largely unstructured but the oligomers display
order dependent increase in β-sheet content (Ono, Condron, & Teplow, 2009).
These are detected as intense species in the brains in Alzheimer’s patients.
Neurotoxic activity increases with oligomer order in these patients (Ono et al.,
2009). They are thought to play a role in impairment of synaptic plasticity, and this
is mediated by cellular prion protein (Laurén, Gimbel, Nygaard, Gilbert, &
Strittmatter, 2009).
In case of our project, we are looking at the N terminus domain of Orb2A
isoform as the domain that appears to initiate the oligomer formation process.
Another similar example is Sup35p, which is shown to form largely unstructured
soluble oligomers. These rapidly convert to species with extensive β-sheet
structure and such conversion is facilitated by disulfide links forming in the N
terminus of the peptide (Chiti & Dobson, 2006).
Our collaborator research group made observations which suggest the
importance the N terminus of Orb2A in regulating the oligomerization process and
thus in turn the aggregation of Orb2. A point mutation (F5Y) and deletion mutant
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(deletion of first 8 N-terminus amino acids - ∆2-8) in Orb2A inhibits long term
memory formation in drosophila (Majumdar et al., 2012). These mutations are
thought to have an effect on oligomer formation characteristics of Orb2A.
Another example about the early stage oligomer formation regulated by N
terminus helix is shown by observations made for Huntingtin protein. Here, 17
amino acid N- terminal segment of Huntingtin fragment (htt) which dramatically
increases the aggregation rate and changes the mechanism of aggregation as
compared to only Poly Q peptide of similar length (Jayaraman et al., 2011). This
leads to rapid formation of α-helix rich oligomeric species. As the aggregation
proceeds, β-sheet structure is slowly strengthened. Higher order α-helix rich
oligomeric structure appear to build up via initial tetramer form. Although, the
peptides form α-helix rich oligomers, only peptides with Q
N
with N=8 or more,
mature into a β-sheet rich fibrils. The final amyloid like aggregates do not only
feature β-sheet rich structure, but also retain some solvent exposed α-helical
structure. The α-helix rich oligomers can be both on and off-pathway. Some
contribute to formation of nuclei while the rest provide for the elongation process.
The existing evidence indicates that these oligomers associate via a four-helix
assembly unit. The poly Q expansion contributes towards enhancing the rates of
oligomer formation and nucleation step, but the structural mechanism is yet
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unclear (Jayaraman et al., 2011; Mishra et al., 2012; Sivanandam et al., 2011;
Wetzel, 2012).
Thus, considering the variation and degree of polymorphism seen in case of
oligomeric species, it is interesting for us to investigate the oligomer formation
process and the regulatory mechanism for Orb2. Such studies would provide a way
to understand the structural mechanism of conversion from oligomeric to extensive
β-sheet rich mature fibrillar structure. Also, one of the long term aims is to
elucidate the role of oligomeric species with regards to nucleation process and how
they can reduce the lag phase in context of functional amyloids.
Long Term Memory Formation
A long standing question regarding long term memory maintenance has been
how the memory trace is persisted when the proteins that initiate the process turn
over and degraded within days.
Learning changes the efficiency and number of synaptic connections. This
includes qualitative and quantitative changes in terms of local protein composition
of synapse. Synthesis of new protein at synapse is important for stabilizing the
functional changes physical growth of synapse important for memory formation
(Darnell & Richter, 2012; Majumdar et al., 2012; Si, Giustetto, et al., 2003).
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CPEB Protein Family is required for Stabilizing Activity Dependent
Synaptic Changes
A family of RNA binding proteins CPEB, specifically working at synapse,
has been identified as the regulator for activity dependent synthesis of proteins. In
Aplysia Californica (Marine Snail), a neuron specific variant of CPEB, ApCPEB is
required not for initial synaptic efficacy or growth following serotonin stimulation
but for maintenance of these changes beyond 24hr (Si et al., 2010). In Drosophila,
reduction in Orb2, a member of CPEB family, prevents memory persistence
beyond 12hr but does not affect short term memory formation (up to 3hrs)
(Majumdar et al., 2012; White-Grindley et al., 2014; Xu, Hafer, Agunwamba, &
Schedl, 2012). In mice, deletion of CPEB-1 gene reduces long term potentiation
triggered by theta-burst stimulation and growth hormone application (Chiti &
Dobson, 2006). These observations suggest an important role of CPEB in long
term stabilization of activity dependent synaptic modifications.

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Mechanism of CPEB Dependent Long Term Memory Formation

Figure 2. Model of CPEB Activity in Amyloid Form Amyloid polymerization of
prion domain CPEB-Q releases sequestered m-RNA binding C terminus of CPEB
for function, results in high local concentration of CPEB and scaffolding and
recruitment of factors involved in translation activation (mRNA with CPEs) Taken
from (Heinrich & Lindquist, 2011a).

The proposed model for the mechanism of CPEB in translation regulation at
synapse is illustrated in Figure 2. The neuronal version of CPEB localizes in
presynaptic terminal of Aplysia and dendrites of mice, and can undergo activation
following synaptic stimulation by neurotransmitter. They bind to U-rich cytosolic
polyadenylation elements (CPEs) in mRNA 3’-UTR and subsequently recruit
polyadenylation machinery. Active CPEB elongates poly-A tails of CPE-
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containing mRNA, which encodes structural and regulatory proteins that is
essential for long term memory formation. Antibody that recognizes oligomeric
ApCPEB selectively blocks persistence of long-term facilitation of sensory-motor
neuron synapse beyond 24hr (Heinrich & Lindquist, 2011b; Huang et al., 2006; Si
et al., 2010; Si, Giustetto, et al., 2003).
In Drosophila, CPEB homologue Orb2 is required for long term
conditioning of male courtship behaviour. Both ApCPEB and Orb2 carry an N
terminal glutamine rich sequence. Deletion of this sequence impairs long term
memory formation indicating important physiological role of prion domain like
sequence (Majumdar et al., 2012). In humans, a particular member of CPEB
family, CPEB3, has been linked to episodic memory formation, suggesting a
conserved role of CPEB in synaptic plasticity and memory (Chiti & Dobson,
2006).
Stimulation of behaviourally relevant neurons increases the levels of
amyloid-like aggregates of Orb2, which is enriched in synaptic membrane fraction.
These observations lead to suggestion that amylogenic conversion of CPEB is
conserved across the species and indeed at to stabilize activity-dependent increase
in synaptic efficacy.
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Fgure3. Role of Orb2 in Long Term Memory Formation Orb2 undergoes a
dopamine dependent aggregation and binds to dormant mRNA leading to local
protein synthesis at synapse essential for long term potentiation. (Taken from
(Bailey, Kandel, & Si, 2004)).
Domain Structure of Rare Isoform Orb2A and Predominant
Isoform Orb2B
The Drosophila Orb2 gene has two protein isoforms, Orb2A and Orb2B and
the oligomers are composed of both Orb2A and Orb2B. In the adult brain, Orb2A
is expressed at very low levels as compared to Orb2B. The domain structure of
Orb2A and Orb2B is described in Figure 4, in which a glutamine rich domain, a
glycine rich domain, two RRMs and Zn
+2
fingers are highlighted for both the
proteins. The two isoforms differ in their domain structure with respect to their N
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terminal domain (Li & Dubnau, 2012; Majumdar et al., 2012). The glutamine rich
domain (prion-like) provides a switch for conversion from soluble to amyloid
genic aggregated state under synaptic stimulation by neurotransmitter (Majumdar
et al., 2012). It’s thought that the N-terminus of Orb2A forms an amphiphillic
helix, which initiates the oligomerization of protein. This oligomerization of
Orb2A is critical for oligomerization of Orb2B. The role of the N-terminus
amphiphillic helix and Orb2A oligomers in the overall process of amyloid
formation of Orb2 is further illustrated in subsequent sections where the hypothesis
is presented.

Figure4. Domain Structure of Orb2A and Orb2B Q- Glutamine rich domain, G-
Glycine rich domain, RRM- RNA recognition motif, Zn- Zn
+2
fingers. The
proposed amphiphillic helix is illustrated for Orb2A and the residues whose
mutations is critical for long term memory formation are marked in red.

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Central Working Hypothesis about Orb2 Amyloid Formation

Figure 5. Central Working Hypothesis for Orb2 Functional Amyloid
Formation. An N terminal amphiphillic initiates helical oligomer formation of
aggregation starter Orb2A, and the glutamine/histidine rich domain forms the
amyloid core. Orb2B is seeded by Orb2A. The C terminus RNA binding domain
and Zn finger do not participate in amyloid core but are responsible for translation
regulation activity by binding to 3’ UTR of mRNA.
Despite low abundance, Orb2A is critical for oligomerization of Orb2 and
Orb2A forms oligomers readily as compared to Orb2B. A mutation highlighted in
red (at F5 in proposed amphiphillic helix of Orb2A) in Figure 4 has been shown to
adversely impact persistence of memory in drosophila (Majumdar et al., 2012).
The prion like domain of Orb2A is sufficient for long-term memory formation. The
hypothesis presented in Figure 5 is in line with the observations made in context of
Page | 17

Huntingtin protein oligomer formation and the importance of N terminal helix in
regulation of oligomer formation process.

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Objectives of the Project
As presented in the hypothesis regarding the Orb2 aggregation, we aim to
test the early stage oligomer formation of aggregation initiator Orb2A.
Orb2A aggregates more readily as compared to abundantly present isoform
Orb2B. The aggregation of Orb2A is also shown to be important for formation of
functional amyloid formation of Orb2. Thus, we propose that Orb2A aggregates
through formation of precursor stage soluble oligomers, which facilitate in
overcoming the kinetic barrier to rapidly form the amyloid fibre nucleus essential
for physiological role of the mature amyloid fibres.
The objectives for this project are: 1. Establish oligomer formation of the
aggregation initiator protein isoform Orb2A, 2. Characterize size, shape and
secondary structure the early stage oligomers, 3. Establish the kinetics of
aggregation of Orb2A and the role of oligomers in the kinetics.
Aim of my research is also to establish a gel based method to detect and
characterize early stage oligomers during the formation of amyloid fibrils. The
same method can be further used to detect various precursor metastable species
during the fibril formation with required modifications.
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Another issue is presented in the hypothesis is that; previous research data
indicates that N-terminal amphiphillic helix of Orb2A regulates the oligomer
formation and thus in turn the aggregation process. A point mutation (F5Y) and
deletion mutant (deletion of first 8 N-terminus amino acids - ∆2-8) in Orb2A
inhibits long term memory formation in drosophila (Majumdar et al., 2012)
described in introduction section. We aim to test these claims by investigating the
effects of mutations of size, shape, stability and secondary structure of early stage
oligomers.
We would also like to characterize the proposed N terminus amphiphillic
helix by investigating its secondary structure and ability to oligomerize in solution
independently.

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Preliminary Work: Orb2A88 Validly Represents the Amyloid Core
The data presented in this section were gathered previous to my arrival in the
research group.
To focus on N terminal domain of Orb2A and due to lower yield of 551
amino acid Orb2A protein during recombinant expression, it was decided to use a
shorter fragment of this protein. It is important that such shortened fragment can
form mature amyloid fibril and can exactly represent the amyloid core as presented
by full length protein (Orb2A_FL). As the RRM and Zn
+2
domains are not part of
the amyloid fibril core but are involved in RNA binding and translation regulation,
these are removed in making the shortened fragment – Orb2A88. These fragment
is made up of N terminus 88 amino acids with a His tag at C terminus added to
facilitate the purification of the fragment. The validity of the fragment is verified
using following tests
1. ThT Fluorescence Assay: Orb2A88 Can Form Mature Amyloid Fibrils
ThT binds specifically to β-sheet rich structure and fluoresces
indicating the presence of amyloid fibrils. Orb2A88 was allowed to fibrilize
and the presence of fibrils was determined using ThT fluorescence assay.
Enhanced fluorescence by ThT after binding to Orb2A88 can be seen in
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Figure 6 which indicates that this shortened fragment can form mature
amyloid fibres.

Figure 6. Orb2A88 can form mature amyloid fibres. Increased fluorescence by
ThT following binding to Orb2A88 as compared to baseline control (buffer) is an
indicative of presence of β-sheet rich mature amyloid fibres.

2. Orb2A88 is Representative of Amyloid Core
Orb2A88 can for β-sheet rich amyloid fibres, but it is important to
verify if Orb2A88 amyloid fibre core is identical to fibre core of Orb2A_FL.
The amyloid fibril core is static in nature. 1D
13
C CP MAS Solid State
NMR spectra are sensitive to this static amyloid core. The strikingly similar
spectra measured for the Orb2A88 and Orb2A_FL can be seen in Figure 7.
Page | 22

This shows the identity of the amyloid cores formed by shortened peptide
and the full length protein.

Figure 7. Orb2A88 amyloid core is identical to that formed by Orb2A_FL
amyloid fibers. 1D
13
C CP MAS Solid State NMR spectra measured for Orb2A88
and Orb2A_FL show strikingly similar nature.
Constructs
Based on the preliminary data and the working hypothesis, following
constructs were produced to study the role of N-terminus in oligomer formation
and study the effect mutations described in objectives section.
MYNKFVNFICGGLPNLNLNKPPQLHQQQ…GGSLEHHHHHH*-Orb2A88_WT
MYNKY VNFICGGLPNLNLNKPPQLHQQQ…GGSLEHHHHHH*-Orb2A88_F5Y
MYNKFVNFICGGLPNLNLNKPPQLHQQQ…GGSLEHHHHHH*-Orb2A_9-88
MYNKFVNFICGGLPNLNLNKPP -Orb2A_1-22
(Orb2A_1-22 is a synthetic peptide)
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Results
UV Scattering Indicates Presence of Early Stage Soluble Oligomers

Figure 8. Scattering of UV light by Orb2A fragment is indication of presence
of oligomers. Orb2AWT, Orb2A88_F5Y and Orb2A_9-88 scatter UV radiation
post renaturation (buffer exchange from 8M urea buffer to 1M urea buffer). All the
proteins are tested after normalizing the concentration at 0.6 mg/ml.
The proteins are produced by overexpressing in Rosetta (E. coli) strain and
purified in 8M urea buffer using a Ni
+2
column. The protein at this stage can be
shock frozen for further use.
Page | 24

The proteins are renatured by desalting from 8M urea buffer to 1M urea
buffer with pH=7, glycerol (10% v/v) and salt concentration (100 mM). UV
absorption characteristic of protein are measured immediately after renaturation,
while the concentration is determined and normalized using Bradford assay.
Proteins containing aromatic amino acids (Tyrosine, Tryptophan, Phenylalanine)
have a maximum absorption peak for UV light at ~280 nm, which can be seen for
Orb2A88_WT and Orb2A88_F5Y, but Orb2A_9-88 does not show this absorbance
as it does not have tyrosine or tryptophan residue. The spectra in Figure 8 shows
all three proteins scatter UV light (250-280 nm) which is not seen for proteins that
remain monomeric in solution.
Similar test was done for Orb2A_1-22 fragment by renaturing the synthetic
peptide (powder) by solubilizing in 1M urea buffer. The spectra shown in Figure 9
does not show scattering of UV light but considering very small size of fragment,
this one test is insufficient to rule out presence of oligomers.

Page | 25

Figure 9. Orb2A_1-22 in 1M urea scatters UV radiation. The absence of
scattering is not sufficient to rule out oligomer presence, as the peptide is much
smaller compared to Orb2A88_WT and the oligomers (if present) could be of
much smaller order.

Orb2A88_WT and Orb2A88_F5Y Form Oligomers in Solution
before Forming Fibres
Having an indication about presence of oligomers by UV scattering, the
oligomer formation in solution by Orb2A88 was confirmed using Dynamic Light
Page | 26

Scattering (DLS). The DLS experiments provided measurements regarding the size
of the oligomers.
The DLS measurements were done maintaining same conditions as the UV
scattering experiments. The theory for size determination using DLS is described
in methods and materials section. Results from DLS experiments are described in
Figure 10 and Figure 11 as the autocorrelation function and the corresponding fit
which results in size distribution profile of oligomers described as function of mass
% of the scattering molecule.
Orb2A88_WT following renaturation, is almost entirely present in soluble
oligomeric state with radius measuring r = 125.5 nm. Orb2A88_F5Y forms
oligomers immediately following renaturation with slightly larger size, with radius
measuring r = 148.2 nm. We also wanted to see the effects of 0M urea buffer and
these tests were done for Orb2A88_F5Y, which forms very large oligomers. But
these oligomers do not have a consistent size distribution and the protein forms
non-specific aggregates and falls out of the solution. DLS experiments for
Orb2A_9-88 and Orb2A_1-22 did not give stable readings.

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Figure 10. Orb2A88_WT and Orb2A88_F5Y form oligomers in solution
immediately after renaturation. Size distribution of the oligomers is shown in
right hand side graph with calculated radius (125. 5 nm for Orb2A88_WT and
148.2 nm for Orb2A88_F5Y). The radius is calculated with predicted shape of
oligomers as isotropic spheres. Left hand side panel shows the autocorrelation
function decay curve. Red line is the theoretical prediction of decay and the blue
line shows the actual experimental measurement. Almost perfect fit of theoretical
and experimental autocorrelation function decay is an indicative of validity and
reliability of the experimental results.

Page | 28

Figure 11. Orb2A88_F5Y in 0M urea forms very large oligomers. The size
distribution of oligomers is not homogenous as seen in right hand side panel. Also,
the autocorrelation function decay is much prolonged due to presence of large
scattering molecules. Though, protein in 0M urea buffer is not very stable and falls
out of solution.

Early Stage Soluble Oligomers are Imaged Using Transmission
Electron Microscopy (TEM)
DLS experiments confirm the presence of oligomeric species immediately
after renaturation process. These oligomers would be visualized using
Transmission Electron Microscopy (TEM). The grids for EM were made using
same protein sample as used for UV spectroscopy experiments with 1:50 dilution
from initial concentration of 0.6 mg/ml.
The oligomers can be seen as well defined, negatively stained species. Such
oligomers can be observed for both Orb2A88_WT and Orb2A88_F5Y as shown in
Page | 29

the Figure 12. These oligomers appear to be spherical shaped with diameter
~34nm as seen by electron micrographs.

Figure 12. Electron micrographs show presence of early stage oligomers. Panel
A and Panel B show oligomers of Orb2A88_F5Y and Orb2A88_WT respectively.
Oligomers observed by TEM were well defined, negatively stained spherical
species.

Developing Semi Denaturating SDS – Agarose Gel Electrophoresis
to Detect Early Stage Oligomeric Species
A key characteristic of amyloid fibres is their resistance to denaturation by
2% SDS at which non amylogenic proteins are denatured. This allows to
differentiate between amyloid and non fibrillar species on a partially denaturating
gel. But native poly acrylamide gel electrophoresis (native PAGE) is inadequate to
detect these very high molecular weight amyloid fibres. Thus, semi – denaturating
Page | 30

agarose based gels are used which are able to detect these amyloid fibres
(Halfmann & Lindquist, 2008). The precursor species though are dissolved or
denatured by 2% SDS concentration. The SDS resistance characteristic increases
as the fibrils mature. Thus, there is a need for conditions which do not break early
stage oligomers as well as differentiate them from monomeric form.
Controls
Two different types of sample preparation systems are used here. A regular
SDS – PAGE loading buffer (2% SDS, β – mercaptoethanol) + boiling at 95
°
C
treatment is used to completely denature protein and represent monomeric form.
To detect the early stage oligomers, the loading buffer used here had a low SDS
concentration (0.01%) enough to provide negative charge for migration under
electric field. A non-reducing environment is maintained as the disulfide links
might be important to oligomer formation. Along with almost native like loading
conditions, the samples are not boiled but only allowed to stand in loading buffer at
room temperature for 10 min. These are non – denaturating conditions. The
samples are run on semi denaturating SDS – Agarose gels made with 1.5% agarose
and 0.01% SDS concentration in gel. Following controls show that these
conditions are valid and differentiate oligomeric state from monomeric form based
on molecular mass and size.
Page | 31

On a denaturating PAGE, the protein irrespective of any of the two loading
conditions (completely denaturating and non denaturating), must be in monomeric
form. This is seen in Figure 13, where the monomeric bands of Orb2A88_WT,
Orb2A88_F5Y and Orb2A_9-88 are detected parallel to each other. Due to non-
reducing environment under non denaturating conditions, fraction of protein is
present as disulfide dimers represented by slow migrating band. This shows that
the migration of protein under voltage difference is not affected by different
loading conditions. So the concern that lower SDS concentration may result in
slower is addressed in Figure 13.

Figure 13. Migration of protein under voltage difference is unaffected by
loading conditions. Under denaturating PAGE, proteins run as monomeric bands
irrespective of different treatments prior to loading on gel. Presence of disulfide
dimers is seen under non denaturating condition due to non-reducing environment.

Page | 32

The two loading conditions are also tested on a semi denaturating agarose
gel for a BSA (2 mg/ml) which does not form large oligomers as Orb2A does.
Though, under non reducing environments the disulfide links are not completely
broken. BSA also forms small oligomers in solution and this is seen by small gel
shift in Figure 14. But the gel shift seen for BSA in Figure 14 is much less as
compared to gel shift seen due to presence of large oligomers in Figure 15. These
two controls together validate the method so that the gel shifts as indicative of
large oligomers are true and not false positives due to non denaturating conditions.

Figure 14. BSA detected under complete denaturating and non denaturating
condition. Small gel shift seen from monomeric form of BSA (Complete
denaturation) is due to formation of small oligomers.

For the detection of oligomers, various staining techniques were tried to get
a better signal to noise ratio. The challenge here is that these oligomeric or fibrillar
species under non denaturating condition appear as smear rather than a thin band
Page | 33

for monomeric condition. Due to this the local protein concentration on gel drops
reducing signal to noise ratio.
These proteins can be viewed by coomassie staining but the detection is low
and it often fails to detect oligomeric conditions. Also, due to high thickness of gel,
staining and de staining process takes very long time (~24-30hrs in total). Capillary
blotting was also tried to blot protein on a nitrocellulose membrane followed by
detection using Ponceau S staining or anti his tag antibody. But both attempts did
not give satisfactory detection.
Thus, a highly sensitive fluorescent Flamingo stain was used and gave the
best detection of all the tried methods. Figure 15 shows the monomeric and
oligomeric condition of Orb2A88_WT, Orb2A88_F5Y and Orb2A_9-88 is under
completely denaturating and non denaturating conditions respectively. The gel is
stained using flamingo stain and detected under UV illumination.
Page | 34

Figure15. The early stage oligomers are detected using semi denaturating
SDS-Agarose Gel Electrophoresis. The early stage soluble oligomers are
differentiated from the monomeric form on 1.5% agarose gel with 0.01% SDS. The
Protein bands are detected using flamingo fluorescence staining with UV
illumination.
F5Y Mutation may Stabilize the Oligomeric State of Orb2A
To test if F5Y mutation has any effect on oligomeric condition stability,
Orb2A88_WT and Orb2A88_F5Y were run on SDS-agarose gel under varying
degree of conditions described in Figure 16. The tests are carried out here 36hrs
after renaturation with no visual aggregates. The results shown in Figure 15
Page | 35

indicate that Orb2A88_F5Y oligomers over the time have become resistant to SDS
denaturation though no fibres are formed.

Figure 16. Orb2A88_F5Y stabilizes oligomeric state. The loading buffer
conditions are: a. 0.01%SDS+no mercaptoethanol + no boiling, b.0.01%SDS+no
mercaptoethanol + boiling, c. 2% SDS+ mercaptoethanol+ no boiling, d. 2% SDS
+ mercaptoethanol + no boiling.
Secondary Structure Analysis
The CD spectra taken for the proteins after buffer exchange from 8M urea
buffer to a CD compatible buffer are shown in Figure 17. Orb2A88_WT early
stage oligomers show a dominant α helical secondary structure. Though
Page | 36

Orb2A88_F5Y forms oligomers in solution, the mutation disrupts the predominant
helical structure. Orb2A88_F5Y oligomers are largely unstructured. Despite
deletion of 2-8 N terminus amino acids, Orb2A_9-88 secondary structure is
predominantly α helical similar to that of wild type oligomers. An unexpected
result is seen in case of Orb2_1-22 which shows a mixture of secondary structure
though hypothesized to form amphiphillic helix.

Figure 17. Secondary structure analysis shows that Orb2A88_WT and Orb2A_9-
88 early stage oligomers are predominantly α helical. F5Y mutation disrupts the
helical nature of early stage oligomers. Orb2A_1-22 does not display a pure helical
structure but forms mixture of secondary structures.

Page | 37

Discussion
Previous studies have shown that in Drosophila adult brain, at physiological
concentration Orb2 forms amyloid like aggregated structures. Upon stimulation of
behaviourally relevant neurons, amyloid aggregation of Orb2 is increased and such
are enriched in synaptic membrane fractions. The aggregates stabilize the activity-
dependent increase in synaptic efficacy (Majumdar et al., 2012). Long term aim of
our lab is to understand the mechanism and regulation of the amyloid fibre
formation of Orb2. We believe as presented in the hypothesis, the Orb2
aggregation process is initiated by rare isoform Orb2A which aggregates readily.
We also propose that Orb2A aggregates through formation of soluble oligomers
which facilitate in nucleation and amyloid core formation process. The process of
oligomer formation is regulated via N terminus amphiphillic helix.
We find that Orb2A88_WT, Orb2A88_F5Y and Orb2A_9-88 scatter UV
radiation after the proteins are renatured by buffer exchange (8M urea to 1M urea
buffer). Rayleigh’s criteria for scattering intensity by molecules is given by
I=I
0
(1+cos
2
Ɵ)8π
4
α
2
/λ
4
R
2
. One of the requirements for scattering is that the
scattering particles must be of the order comparable to that of the wavelength of
incident radiation. The observations described in Figure 8 thus indicate presence of
molecules with size of the order of UV radiation wavelength, and thus presence of
Page | 38

the proteins may be forming oligomeric species. At the same time, we also
observed that Orb2A_1-22 does not scatter UV light. This is not sufficient to say
that Orb2A_1-22 does not oligomerize in solution. The peptide is very small and
thus it might form oligomers in solution but of much smaller order compared to
that of UV radiation wavelength, and thus no scattering is seen. The presence of
oligomeric species for Orb2A88_WT and Orb2A88_F5Y was confirmed by DLS,
but Orb2A_9-88 and Orb2A_1-22 did not provide stable readings. DLS
experiments predict the radius of oligomers measured as isotropic spheres 125.5
nm and 148.2 nm for Orb2A88_WT and Orb2A88_F5Y respectively. The size of
this oligomers (d = 250-280 nm) is of the order of UV wavelength and thus the
data correlates well from both these experiments. The data also highlights that
Orb2A88_WT and Orb2A88_F5Y are almost entirely present in solution as
oligomeric species. The oligomers form very rapidly and are much larger than
some of the oligomers reported in the literature(Chiti & Dobson, 2006; Jayaraman
et al., 2011). The possible explanation for this is that due to physiological role of
the protein, the amylogenic conversion upon activation must occur in a short
specified time. The rapidly forming soluble oligomers of Orb2A may facilitate to
overcome this barrier for rapid association and initiation of nucleation and amyloid
core formation. Thus, monomeric form of the protein post renaturation is rarely
present. Entire protein in solution is in oligomeric form and the fraction is also
Page | 39

largely homogenous as far as the size of oligomers. These early stage oligomers
are visualized by TEM as spherical shape, negatively stained species with ~ d = 34
nm. This is much different than the size determined by DLS. This could be due to
staining with 1% uranyl acetate, compression of 3D structure and lateral diffusion
while making the EM grids. The grids are also made with diluting the protein
concentration 1:50 from starting concentration of 0.6 mg/ml which is used for DLS
experiments.
The resolution of amyloid aggregates based on size and detergent
insolubility has been made possible with the use of semi denaturating SDS –
agarose gel electrophoresis. The SDS resistant amyloids can be resolved from
proteins that are denatured by 2% SDS concentration as present in normal SDS –
PAGE. Early stage oligomers though will be denatured or dissolved by 2% SDS
concentration. We modified the existing SDS-AGE method by creating two
different loading conditions, one to denature the protein sample to monomeric state
while other to maintain the protein in native condition (non denaturating). Thus if
the amylogenic protein forms soluble oligomers, they will be seen under non
denaturating condition and can be resolved from their monomeric form based on
gel shift as seen in Figure 15. The SDS-AGE used to resolve amyloid aggregates
detects them as large smears formed on the gel as shown in Figure 18. Contrary to
this, we detected monomers and early stage oligomers as sharp bands or small
Page | 40

blobs which can be seen in Figure 15 and 16. In the future, the method can be
improved by devising a way to compare size and molecular mass of the oligomeric
species by their position on the gel.

Figure 18. The presence of very large molecular weight amyloid aggregates is
detected by semi denaturating SDS-AGE. The amylogenic aggregates are
detected as large smears as marked in the figure, whereas the non amylogenic
proteins are solubilized/ denatured by existing condition and thus migrate farther
on the gel (Taken from (Halfmann & Lindquist, 2008)).

Analysing the fate of oligomeric state 36hrs after renaturation with no visual
aggregates, we found that Orb2A88_F5Y still in oligomeric state had become
resistant to SDS denaturation treatment. This is a very preliminary indication that
Orb2A88_F5Y mutant may strengthen the oligomeric state and prevent the
Page | 41

transition to amyloid like aggregates essential for formation of mature and
functional Orb2 fibrils.
Analysing the secondary structure of these oligomers, we observed that
Orb2A88_WT forms predominantly α-helical structures. This is in accordance
with one of the proposition of working hypothesis that Orb2A rapidly forms α-
helical oligomers in solution. A model has been prosed for another amyloid protein
Huntingtin, which also forms such oligomers. In this model, the N terminus
segment is engaged immediately in a stable α-helix after oligomer assembly
initiates. At least a noticeable portion of α-helical characteristic is maintained
throughout nucleation and elongation process. The N terminus being assembled
into helical core, as these assembly grows, it increases local Poly Q segment
concentration, thus facilitating nuclei formation. The elongation of the amyloid
aggregates can occur by addition of oligomers itself to the nuclei (Chiti & Dobson,
2006) but a for Huntingtin aggregation, it’s thought to occur by addition of
monomers. These monomers are provided by dissociation of oligomers that do not
participate in nucleation (Jayaraman et al., 2011; Mishra et al., 2012; Scherzinger
et al., 1999). In case of Orb2A, we believe a similar model could be working as
proposed in the hypothesis. Orb2A88_F5Y mutant disrupts this predominant α-
helical structure of the oligomers. If the proposed model is correct, then non helical
F5Y mutant oligomer may prevent the nucleation and elongation process. We
Page | 42

expected that deletion of 2-8 amino acid residues would disrupt the helical
structure and may even impair oligomer formation capacity of fragment, but
Orb2A_9-88 retains the helical characteristic. This fragment though tends to
aggregate rapidly and fall out of solution. These aggregates could be non-specific
in nature and need further examination. The yield after purification is also lower
though it is not clear if the overall expression is low or the purification
characteristics are altered as compared to Orb2A88_WT and F5Y. The secondary
structure shown by Orb2A_1-22 peptide in isolation is a mixture of secondary
structures and not a purely α-helical structure as initially proposed.
These observations together indicate that the N terminus is important for
oligomer formation as a point mutation in this region disrupts the helical nature of
oligomers. Containing large number of hydrophobic amino acids, Orb2A_1-22 is a
candidate for forming an amphiphillic helix, but the helicity might be induced by
some inter or intra molecular interactions or other factors.
We propose another mutant with deletion 22 N terminus amino acids (Entire
proposed amphiphillic helix) and check the effect of such mutation on oligomer
formation and secondary structure of Orb2A. A comparative analysis of
aggregation kinetics of Orb2A88_WT with F5Y and other mutants would give an
estimate about length of nucleation step and the effect of mutations on the length
Page | 43

of nucleation. Our lab is also currently investigating metal binding characteristics
of Orb2A. It will be also interesting to investigate interaction of protein with
metals, lipids or other molecules and its effect on oligomer formation.

Page | 44

Materials and Methods
DNA cloning, Transformation, Protein Expression, Purification and
Renaturation (Desalting)
1. Cloning: We used pET28b vector containing T7 promoter, chloramphenicol
and kanamycin resistant genes. Orb2A88_WT sequence is cloned into the
vector using Xho1 and NcoI restriction enzyme. Mutant Orb2A88_F5Y was
generated QuickChange II XL Site – Directed Mutagenesis Kit whereas
Orb2A_9-88 was cloned as Orb2A88_WT. Xl 10 gold ultra-competent cells
were transformed with vector and selection was done using kanamycin
resistance. Zyppy
TM
Plasmid Miniprep Kit was used to purify plasmid from
the overnight culture (25 ml) of transformed cells.
2. Transformation: Rosetta Bl21 (DE) chemically competent cells were
transformed using heat shock (42°C water bath for 45 sec) and successfully
transformed cells were selected using chloramphenicol and kanamycin
resistance.
3. Protein Expression: Transformed Rosetta culture was grown in 25 ml LB
(chloramphenicol + kanamycin) overnight at 37°C. The overnight culture
was used as starting culture for 500 ml LB (chloramphenicol + kanamycin)
culture. The cells were induced once the OD was reached at 0.6 using IPTG
Page | 45

and protein was expressed for ~4-5 hrs. The cells were spun down @ 3500
RPM for 20 min and the pellets were stored at -80°C for protein purification.
4. Protein Purification:
(Buffers: 8M urea, 250mM NaCl, 100mM Dibasic Phosphate, glycerol 10%
(v/v), protease inhibitor, pH=8 (for pellet re-suspension, Ni
+2
column
equilibration and washing), pH=6.75 (Ni
+2
column washing ~50 ml), pH=
5.25, 4.75,4.25,3.75 (protein elution ~10ml each). Orb2A88 is eluted largely
in pH=4.75 or 4.25.*)
The cell pellet is re-suspended in 8M urea pH=8 buffer (~25ml) Cells are
sonicated 1.5 min 3 times at medium/low power level keeping the solution
on ice. Cell lysate is centrifuged at 20000 RPM for 20 min and the
supernatant is placed on equilibrated Ni
+2
column. The protein is eluted
using pH gradient elution and the purity is verified by SDS-PAGE. Elution
containing our protein is fractioned in 2ml fractions and shock frozen and
stored at -80°C for future use.
5. Renaturation (Desalting):
GT Buffer- 1M urea, 10mM HEPES, 100mM KCL, 10% glycerol (v/v),
pH=7 (used for UV spectroscopy, DLS, SDS-Agarose electrophoresis and
EM)
Page | 46

For CD spectroscopy (secondary structure analysis): 75mM Tris, 10mM
NaCl, pH=7.6
PD 10 desalting column is equilibrated using appropriate buffer. Maximum
of 2.5ml of protein elution fraction (in 8M urea) is placed on the column.
Protein is eluted from the column using 2.5ml GT buffer/buffer for CD.
Synthetic Peptide:
Orb2A_1-22 (MW 2493.1g/mol) was ordered as synthetic peptide and not cloned
in lab. The peptide is purchased from Eurogentec in dried format and is dissolved
in appropriate buffer (GT buffer/buffer for CD) prior to experiments.
UV spectroscopy
After renaturation, protein concentration is determined using Bradford Assay and
is normalized to 0.6 mg/ml for experiments for all protein (Orb2A88_WT,
Orb2A88_F5Y and Orb2A_9-88). The spectrum (in triplicates) is recorded on
Agilent Cary 60 UV VIS in-house spectrometer for each protein. VWR quarz
spectrophotometer cell (10mm light path length) was used and the scans were
recorded using inbuilt slow scan mode. The data is plotted using Matplotlib.

Page | 47

CD Spectroscopy
CD spectra were obtained using Jasco J-810 spectropolarimeter in Dr. Ralf
Langen’s lab with 1mm quartz cell at room temperature. A scan rate of 50nm/min,
bandwidth of 1nm, and 0.1nm time response and step resolution of 0.5nm was used
for all measurements and blanks. Protein concentration was determined using
Bradford assay and all proteins were normalized at 0.15mg/ml for measurements.
Electron Microscopy
Samples were negatively stained for transmission electron microscopy studies.
Carbon-coated formvar mounted on copper grids were floated on 50µl droplet
(1:50 dilution from starting 0.6 mg/ml) of sample for 5min and excess liquid was
removed from grids using filter paper. The grids were then stained with 1% uranyl
acetate. A JEOL 1400 transmission electron microscope accelerated to 100kV was
used for observation.
Dynamic Light Scattering (DLS)
DLS measurements were carried out on Wyatt DynaPro Nanostar light scattering
instrument in Dr. Janet Oldak’s lab at USC. Total of 10 measurements were
recorded for each sample with each measurements consisting of 10 individual
scans. Dynamic 7 software provided by Wyatt technology provided with fitting of
Page | 48

correlation function and size estimated. Autocorrelation function and size
distribution were plotted using MatPlotLib script.
The size distribution profile measured by DLS is determined from the
autocorrelation function. Second order correlation function is given by

which is generated from the intensity curve. As seen in the
formula, short time delays represents high correlation. This is because the particles
do not have a chance to move much from the initial state that they were in. The
two signals are thus essentially unchanged when compared after only a very short
time interval. As the time delays become longer, the correlation decays
exponentially, meaning that, after a long time period has elapsed, there is no
correlation between the scattered intensity of the initial and final states.
This exponential decay is related to the motion of the particles, specifically to the
diffusion coefficient.
For mono disperse samples first order autocorrelation function is

Translational diffusion coefficient D
t
is calculated by relation ∆t= q
2
D
t
.
The function is described by .
Where q= 4πn
o
sin(Ɵ/2)/λ.
Page | 49

D
t
is used to calculate hydrodynamic radius of particle using Stokes-Einstein
equation D=K
B
T/6πηr.
SDS-Agarose Gel Electrophoresis
Gel Preparation: Standard equipment for horizontal DNA electrophoresis is used.
We prepared gel in 7cm.6cm casting tray with 8 well comb assembly. The gels
were prepared by 1.5% agarose solution in 1X TAE. The volume (~60ml) should
be enough to completely submerge the comb teeth so that maximum volume of
sample can be loaded to maximize detection. The mixture was microwaved until
agarose is completely dissolved. SDS is added rapidly from 10% stock to make the
final concentration 0.01% (the concentration can be increased to 0.1% if the aim is
to resolve amyloid fibrils). The mixture is then quickly poured in the casting tray
uniformly without any bubbles being allowed to form.
Sample preparation and Gel Run
Protein samples are made using two different loading conditions, 1. Non
denaturating - to detect presence of early stage oligomeric species, 2. Complete
denaturation – to represent monomeric state of protein against which oligomeric
state can be compared based on gel shift.
Page | 50

Non denaturating sample preparation: Loading buffer used for this is a 4X sample
buffer (2X TAE, 20% glycerol Bromophenol blue). Β-mercaptoethanol is not
added. The SDS is added before mixing with protein sample such as the final SDS
concentration of loading sample is 0.01%. The sample is not boiled but kept at
room temperature for 10 min before loading on the gel.
Complete denaturating sample preparation: Loading buffer used for this purpose is
same as SDS-PAGE sample buffer (5X buffer – 10% w/v SDS, 10 mM β-
mercaptoethanol, 20% glycerol, 0.2 M Tris-HCl pH 6.8, 0.05% Bromophenol
blue). The samples are boiled at 95°C for 10 min to completely denature the
protein.
The gel was run at low voltage (3 V/cm of gel length) until the dye front reached
~1 cm from the end. This takes several hours and thus the gel is run on ice or in
cold room in order to prevent diffusion of proteins.
Detection
Various detection methods were tried to get maximum detection. We found
Flamingo fluorescent staining worked best among all the methods used and the
results of this are described in Results section. All the methods attempted are
described in this section.
Page | 51

1. Coomassie Staining: SDS-Agarose gel can be stained like a normal
polyacrylamide gel using coomassie staining. Electrophoresis was stopped
and the gel was briefly rinsed with water and submerged completely in
coomassie stain followed by destaining with 20% methanol, 10% acetic acid
solution. The staining and destaining takes a long time (~ 36hrs) due to high
thickness of gel.
2. Blotting: Proteins were blotted on a nitrocellulose membrane using
downward capillary blotting method. Whatman ™ TurboBlotter ™ was used
for this. 20 pieces of GB004, 4 pieces of GB002, one piece of pre wet
GB002 (in TBS), pre wet nitrocellulose membrane are assembled in a stack
in this order. The gel is briefly rinsed and placed on the nitrocellulose
membrane. Trapped bubbles are removed by applying buffer. Three pieces
pre wetted GB002 are placed on the gel. Pre wetted wick was placed across
this stack with either end of the wick submerged in TBS container.
Additional weight for transfer was provided by placing a plastic tray on top
with 150ml bottle. After the transfer, we processed the membrane by
standard western blotting using anti His tag antibody or by staining the blot
with Ponceau S stain. Unfortunately, the detection was not satisfactory with
either method.
Page | 52

3. Flamingo Staining: The gel was briefly rinsed with water and put in a fixing
solution (40% (v/v) ethanol, 10% (v/v) acetic acid) and placed on shaker
inside a cold room overnight. The fixing solution was removed and gel was
put in 50ml of 1X flamingo fluorescent (diluted from 10X stock) and
allowed to stain for at least 3hrs. The container was wrapped in aluminium
foil to avoid exposure to light. The gel was imaged using UVP BioSpectrum
® 500 Imaging system with LM-26 BioChemi 500 camera. Epi illumination
365/480 nm, trans illumination using UV with Chemi filters settings were
used to capture images. The exposure time between 15 to 30 sec works best
for detection. An auto scan series of exposure time was done and the best
image was selected.

Page | 53

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Abstract (if available)

Abstract Amyloid proteins are associated with protein misfolding and aggregation diseases but also many functional amyloid proteins are discovered with physiological role. During the process of amyloid fibril formation, non fibrillar oligomeric species are seen. They are thought to play an important role in nucleation process and amyloid core formation. The focus of our project is the functional amyloid protein Orb2 which plays an important role in long term memory formation in Drosophila. We propose an amphiphillic helix at the N terminal which regulates the oligomer formation and aggregation of Orb2 isoform A. We found that Orb2A rapidly assembles into soluble oligomers measuring ~125 nm radius. These oligomers are predominantly α‐helical secondary structure. A point mutation in the N terminal region of Orb2A (F5Y), which prevents long term memory formation in Drosophila, disrupts the helical structure of oligomers and appears to stabilize oligomeric state. The N terminal 22 amino acid peptide forms a mixture of secondary structure thus suggesting involvement of other domains or external factors in helicity of early stage oligomers. We developed to semi denaturating SDS‐agarose gel electrophoresis method to resolve early stage soluble oligomeric species.

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Asset Metadata

Creator Agashe, Ninad D. (author)

Core Title Oligomer formation of functional amyloid protein - Orb2A

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 08/11/2014

Defense Date 06/17/2014

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

Tag amphiphillic helix,Amyloid,long term memory,OAI-PMH Harvest,oligomers

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Siemer, Ansgar (committee chair), Haworth, Ian S. (committee member), Langen, Ralf (committee member), Tokes, Zoltan A. (committee member)

Creator Email agashe@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-458986

Unique identifier UC11286679

Identifier etd-AgasheNina-2807.pdf (filename),usctheses-c3-458986 (legacy record id)

Legacy Identifier etd-AgasheNina-2807.pdf

Dmrecord 458986

Document Type Thesis

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Rights Agashe, Ninad D.

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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