Structural characterization of the functional amyloid Orb2A using EPR spectroscopy

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Content Structural Characterization of the
Functional Amyloid Orb2A using EPR
Spectroscopy

A Thesis Presented to the Faculty of the
University of Southern California - Graduate School

In Partial Fulfillment of the Requirements
for the Degree of

Master of Science
Biochemistry and Molecular Biology

By: Silvia A. Cervantes
May 2015

August 2015
Acknowledgments:

I would like to thank my committee members, Dr. Ralf Langen, Dr. Zoltan Tokes
and Dr. Ansgar Siemer, for their support and guidance. Their knowledge and insightful
commentary has been invaluable in writing this thesis. I would also like to extend my
gratitude to the members of the Langen Lab for their helpful discussions and assistance
in running my EPR experiments. In particular, I would like to thank Dr. Isas for sharing
his expertise in EPR sample preparation and use of the EPR spectrometer. My sincere
thanks also goes to my lab members for creating a fun and supportive atmosphere in
lab, you guys are the best! I especially would like to extend my gratitude to my PI Dr.
Siemer for his patience and positive attitude, it has truly been a pleasure to be part of
the Siemer lab. I also want to thank the Biochemistry and Molecular Biology Master’s
program for giving me the opportunity to be part of such a great community.
Lastly, but most importantly I want to thank my family, boyfriend and friends for
all their encouragement. I would not be who I am and where I am today without their
unwavering support.
Table of Contents:
1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Introduction
2.1 Amyloid Fibrils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2 CPEB Proteins and Memory Formation . . . . . . . . . . . . . . . . . . . 2
2.3 Functional Amyloid Orb2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.4 Aims of this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Results
3.1 Site Directed Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2 Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.3 Desalting and Spin Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.4 Thioflavin T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
3.5 Lipid Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
3.6 Domain Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
4. Discussion
4.1 Site Directed Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.2 Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.3 Desalting and Spin Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.4 Lipid Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
4.5 Domain Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
5. Materials and Methods
5.1 DNA Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
5.2 Troubleshooting PCR Reactions . . . . . . . . . . . . . . . . . . . . . . . .28
5.3 Transformation and Protein Expression . . . . . . . . . . . . . . . . . 30
5.4 Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
5.5 Spin Labeling and Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
5.6 Thioflavin T Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32
5.7 Lipid Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5.8 EPR Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
5.9 Lipid Interaction Measurements . . . . . . . . . . . . . . . . . . . . . . . 34
5.10 UV Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.11Western Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

. . . . . . . . . . . . . . . . . . . . . . . . . 26
Abstract:

saccharomyces cerevisiae
Drosophila



2. Introduction:
2.1 Amyloid Fibrils:

β



Saccharomyces cerevisiae Drosophila
megalonaster Aplysia californica

saccharomyces cerevisiae

Aplysia californica Drosophila
melanogaster



2.2 CPEB Proteins and Memory Formation:



Xenopus

Aplysia
Drosophila Aplysia ap
Drosophila


2.3 Functional Amyloid Orb2A:

ap

Figure 1: The C terminal and Q rich domain are similar in both isoforms. The difference between them
lies in the N terminal domain, indicating that the structural features underlying this region is what provides
each isoform with its unique role in the mechanism of memory formation.
2.4 Aims of This Work:

in vivo

in vitro

3. Results:
3.1 Site Directed Mutagenesis:

μ
μ



Figure 2 : 0.25M and 0.50M betaine greatly
improved the specificity of annealing for L18C
primers in combination with Initial denaturation:
98ºC 30 sec, denaturation: 98ºC 10sec, T =64ºC
and 25 cycles.

Figure 3 : Use of 0.50M and 1.00M betaine in
combination with initial denaturation: 96ºC 5min,
denaturation: 96ºC 45 sec, and 35 cycles led to
successful amplification of Q51C. Additional
attempts using 0.50M, 1M and 1.5M Betaine with
DMSO were unsuccessful.

μ μ

μ
μ



Figure 4 : Excessive cycling converts PCR products
into high molecular weight fragments. Increasing
the number of cycles can result in increased yield
however, it may also result in fragmentation of the
product and nonspecific amplification.

Figure 5 : Increasing the annealing temperature
failed to improve the specificity of Q26C primers.
The low molecular smears also indicate the
formation of secondary structure preventing
amplification.

μ μ
μ

Figure 6 : The G2Y mutation was successfully
cloned into G12C, Q23C, and Q34C. Low and high
molecular weight smears indicate nonspecific
annealing, however the major product is consistent
with the plasmid weight. Sequencing of products
also confirmed the correct weight and DNA
sequence. The three bands observed in the lane
containing the G12C Y2G plasmid indicate
supercoiling in the plasmid.

Overview of Cysteine Sites for Characterization Using EPR:

1 0 2 0   3 0 4 0 5 0 6 0
MYNKF V NFI C G G LPNLN L NK   PP Q LH Q QQHQ   QQH Q QHQQHQ   Q Q QQLHQHQQ Q LSPNLSALH
7 0 8 0   9 0
HH H QQQQQLR   ESGGS H SPSS PGG G GGGSLE   HHHHHH*

Green sites, except for position 10, indicate locations that were successfully mutated through PCR or that
were ordered from GenScript. Red sites indicate positions for which we designed primers, but did not
proceed to optimize cloning.

3.2 Purification:



Figure 7 : The majority of impurities are removed in the Triton X 100 wash. While all Orb2A88 fractions
are eluted pure, the majority of the protein is found in the 100 mM and 150 mM imidazole fractions.





Figure 8 : The H76C double band is prominently
observed in the whole cell lysate and supernatant
fractions. While some of the H76C protein is
isolated in the 150 mM imidazole elution as a
single band, the major part is isolated as a double
band in the 200 mM imidazole elution.

Figure 9 : Oxidized H76C is highly concentrated in
the whole cell lysate, supernatant and flow
through fractions. Although both the reduced and
oxidized proteins are present in the 200mM
elution, the band representing the oxidized protein
is present at a higher concentration. A “ghost
band” can be observed for oxidized H76C as a
result of overexposure due to high concentration.




3.3 Desalting and Spin Labeling:

β
β

Sample Concentrations After PD10:
Sample: Concentration: Method Used:
V6C 1.19 mg/mL Bradford
WT 1.22 mg/mL Bradford
G12C 0.60 mg/mL UV 280 nm
L18C 0.89 mg/mL UV 280 nm
Q23C 0.35 mg/mL UV 280 nm

Sample:

Concentration:

Method Used:
Q34C 1.50 mg/mL Bradford
Q42C 1.04 mg/mL Bradford
Q51C 1.00 mg/mL Bradford
H76C 1.33 mg/mL Bradford
G84C 0.65 mg/mL Bradford

Figure 10 : Scattering dominates both the pH 7.4 and pH 7.4 (2) curves. There is so much scattering in
these samples, that their absorbance does not start at zero. In contrast, the absorbance for the pH 5.0
curve starts at zero and displays a prominent absorbance peak at 280 nm
3.4 Thioflavin T:




Figure 11 : Samples for 88wt, G84C, V6C and Q51C are positive for fibril formation.

3.5 Lipid Interaction:
Lipid:Protein Interaction N terminus

Figure 12 : The strong interaction between V6C and
the N terminus leads to broadening of the central
line and decrease in amplitude for the side peaks.
Broadening is significant even at 10:1 lipid:protein.
Lipid:Protein Interaction C terminus

Figure 13 : H76C is unaffected by the increase in
lipid concentration, even at 100:1 lipid:protein
peaks appear highly dynamic and do not decrease
in amplitude.



3.6 Domain Characterization:



Orb2A88
Residue Number
Inverse Central Line
Width (Gauss 1 )
V6C 0.1227
C10 0.1155
G12C 0.1091
L18C 0.1965
Q42C 0.2801
Q51C 0.1309
G84C 0.6536

Figure 14 : Inverse line width plot for wild type and mutant sites show greatest immobilization at sites in the
Nterminus. Sites in the Q rich region exhibit intermediate immobilization, while the Cterminus is
completely dynamic.





Figure 15 : The V6C mutant exhibits spin exchange. Reduction in spin label allows us to see a clear
change in the spectra.

Figure 16 : The wild type position exhibits slightly less spin exchange as compared to V6C. The 100%
labeled sample contains a small mobile component that can be observed in the outer peaks.

Figure 17 : Spin exchange is greatly reduced at position G12C. Spectra for the 10% and 100% samples is
almost identical in central line width and amplitude.



Figure 18 : Mutant L18C displays no spinspin interaction. Spectra for 10% and 100% samples are
identical.

Figure 19 : Mutant Q42C 100% and 10% are immobilized and display modest spinspin interaction.

Figure 20 : Q51C is immobilized and displays no spinspin interaction. Signal to noise for this mutant is
greatly affected, especially for the 10% labeled sample.

Figure 21 :spinspin interactions at this location are barely detectable. In addition overall line shape,
amplitude and central line width make this the most dynamic sample.



4. Discussion:
4.1 Site Directed Mutagenesis:



Orb2A88 Q Rich Region: Q23 Q51

C A G C T C C A C C A G C AA C A G C AT C AA C AA C A G C AT C A G C A G C A C C A G C AA C AT C AA C A G C A G
C AA C A G C T C C AT C A G C A C C AA C A G C AA

In addition to the high level of repetitiveness, the Q rich region is further complicated by its high GC content
(52.9%). These two factors contribute to the tendency of this region to associate into hairpins.

4.2 Purification:







β β

4.3 Desalting and Spin Labeling:



β



in vivo

4.4 Lipid Interaction:

in vivo

in vitro




4.5 Domain Characterization:

in vivo









β





5. Materials and Methods
5.1 DNA Cloning:
Parent Construct - Orb2A88_C10M:

PCR Cycling Parameters:
Segment Cycles Temperature Time
1 1 95 ºC 1 minute
2 18 95 ºC 50 seconds
66 ºC 50 seconds
68 ºC 3 minutes
3 1 68 ºC 7 minutes

Reaction Components:

5 L of 10X Reaction Buffer μ
X L (10 ng) dsDNA template μ
X L (125 ng) of oligonucleotide primer 1 μ
X L (125 ng) of oligonucleotide primer 2 μ
1 L of dNTP mix μ
3 L of QuickSolution reagent μ
ddH 2 O to final volume of 50 L μ
Mutants - V6C, Q42C, H76C, and G84C:

μ

PCR Reaction Components:
Component Volume Final Con.
Phusion PCR MM (2X) 25 L μ 1X
Sense Primer (10 M) μ 2.5 L μ 0.5 M μ
Antisense Primer (10 M) μ 2.5 L μ 0.5 M μ
DMSO 1.5 L μ 3%
Template DNA 1 L μ 10 ng
H 2 O 17.5 L μ

PCR Reaction Parameters:
Step Temperature Time
Initial Denaturation 98 ºC 30 sec.
25 cycles 98 ºC 10 sec.
60 ºC 30 sec.
72 ºC 2.48 min.
Final Extension 72 ºC 7 min.
Hold 20 ºC

Mutants - L18C and Q51C:

μ



Q51C PCR Reaction Parameters:
Step Temperature Time
Initial Denaturation 96 ºC 5 min.
35 cycles 96 ºC 45 sec.
72 ºC 45 sec.
72 ºC 2.48 min.
Final Extension 72 ºC 7 min.
Hold 20 ºC

L18C PCR Reaction Parameters:
Step Temperature Time
Initial Denaturation 98 ºC 30 sec.
25 cycles 98 ºC 10 sec.
64 ºC 30 sec.
72 ºC 2.48 min.
Final Extension 72 ºC 7 min.
Hold 20 ºC

Q51C and L18C PCR Reaction Components:
Component Volume Final Con.
Phusion PCR MM (2X) 25 L μ 1X
Sense Primer (10 M) μ 2.5 L μ 0.5 M μ
Antisense Primer (10 M) μ 2.5 L μ 0.5 M μ
Betaine 2.5 15 L μ .25 1.5M
Template DNA 1 L μ 10 ng
H 2 O 2.5 15 L μ

Mutants - G12C, Q23C, and Q34C:



E. Coli

Dpn1 Digestion:

μ
μ
μ μ

5.2 Troubleshooting PCR Reactions:
PCR Additives:

μ

PCR Parameters:

Q23C PCR Reaction Parameters Using Touchdown PCR:
Phase 1 Step Temperature Time
1 Denature 95 ºC 3 minutes
2 Denature 95 ºC 30 seconds
3 Anneal 88 ºC 76 ºC 45 seconds
4 Elongate 72 ºC 1.50 minutes
steps 2 4 were repeated a total of 13 times, starting with 88 ºC and lowering the temp by 1 ºC each cycle
Phase 2 Step Temperature Time
5 Denature 95 ºC 30 seconds
6 Anneal 78 ºC 45 seconds
7 Elongate 72 ºC 1.50 minutes
steps 5 7 were repeated 25 times
Termination Step Temperature Time
8 Elongate 72 ºC 2 minutes
9 Halt Reaction 4 ºC 15 minutes
10 Hold 20 ºC


6x Agarose Loading Dye with SDS:

5.3 Transformation and Protein Expression:
Transformations:
μ

μ

μ

μ

μ

μ
μ

Protein Expression:
μ μ

μ μ

μ β

5.4 Purification:

β

RC

β

β

β



μ

5.5 Spin Labeling and Dialysis:



r ot e i n C onc e nt r at i on r ot e i n V ol um e P
(
m g
m L
)
P m L m g o f P r o t e i n
5.5 μ L o f S p i n L a b e l

5.6 Thioflavin T Staining:

μ

μ μ

μ

5.7 Lipid Preparation:

sn
μ
μ μ



μ


5.8 EPR Sample Preparation:
Fibril Measurements:







5.9 Lipid Interaction Measurements:



μ

μ
μ

Orb2A88 V6C Lipid Interaction
Lipids
(3000 M) μ
Protein
(90 M) μ
Ratio

3 L μ 100 L μ 1:1
30 L μ 100 L μ 10:1
60 L μ 100 L μ 20:1
150 L μ 100 L μ 50:1
210 L μ 100 L μ 70:1
300 L μ 100 L μ 100:1

Orb2A88 H76C Lipid Interaction
Lipids
(3000 M) μ
Protein
(90 M) μ
Ratio

1.5 L μ 50 L μ 1:1
15 L μ 50 L μ 10:1
30 L μ 50 L μ 20:1
75 L μ 50 L μ 50:1
105 L μ 50 L μ 70:1
150 L μ 50 L μ 100:1

5.10 UV Spectroscopy:
Bradford Assay:



μ
μ

UV 280 nm:

μ
ε
5.11 Western Blot:



5. References:

Quarterly Reviews of Biophysics

Future Medicinal Chemistry

Cold Spring Harbor
Perspectives in Medicine

Nucleic Acids Research

The Lancet

PloS One

Neuron

PLoS
Biology

PLoS Biology

Acta Biochimica Polonica

Cell

Cell

Nature Reviews. Neuroscience

Molecular Brain

Biochimica et Biophysica Acta

Nature Neuroscience

Cell

The Journal
of Biological Chemistry

Asset Metadata

Creator Cervantes Cortes, Silvia A. (author)

Core Title Structural characterization of the functional amyloid Orb2A using EPR spectroscopy

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 06/30/2015

Defense Date 06/29/2015

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

Tag EPR spectroscopy,functional amyloid,OAI-PMH Harvest,Orb2A

Format application/pdf (imt)

Language English

Advisor Langen, Ralf (committee member), Siemer, Ansgar (committee member), Tokes, Zoltan A. (committee member)

Creator Email angie.crvts@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-583308

Unique identifier UC11300356

Identifier etd-CervantesC-3527.pdf (filename),usctheses-c3-583308 (legacy record id)

Legacy Identifier etd-CervantesC-3527.pdf

Dmrecord 583308

Document Type Thesis

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Rights Cervantes Cortes, Silvia A.

Type texts

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

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract Functional amyloids are responsible for a variety of physiological processes in a diverse range of organisms. In humans, Pmel17 is involved in the synthesis of melanin, while in saccharomyces cerevisiae Sup35 and Mod5 have been identified as candidates in for providing antifungal protection to this organism. In Drosophila CPEB Orb2 aggregates have been associated with the formation and maintenance of long term courtship memory in adult flies. Previous research has also shown that although Orb2 fibrils are composed of two isoforms, Orb2A and Orb2B, Orb2A is the essential component for the nucleation and regulation of fibrils. Specifically, this work has also revealed that the first 88 amino acids of Orb2A are the residues responsible for the formation and maintenance of long term memory. Although much is known of the role and biological function these fibrils play in stabilizing long term memory, information on the structural features characterizing them is lacking. Here we use site directed spin labeling in conjunction with EPR to examine the underlying structural features of fibrils formed by Orb2A88. In particular we provide EPR analysis of seven sites throughout the Orb2A88 fragment, thereby identifying the static and dynamic domains as well as fibril core of this 88 amino acid protein.