Protein Amyloid Aggregation: Methods and Protocols

317 Pages·2016·10.678 MB·English

by  David Eliezer (eds.)

Methods in
Molecular Biology 1345
David Eliezer Editor
Protein
Amyloid
Aggregation
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire , AL10 9AB, UK
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Protein Amyloid Aggregation
Methods and Protocols
Edited by
David Eliezer
Weill Cornell Medical College, New York, NY, USA
Editor
David Eliezer
Weill Cornell Medical College
New York , NY, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-2977-1 ISBN 978-1-4939-2978-8 (eBook)
DOI 10.1007/978-1-4939-2978-8
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Prefa ce
This volume is focused on methods for the characterization of aggregation processes that
lead to the formation of amyloid fi brils and amyloid oligomers that feature in the etiology
of a variety of human disorders collectively known as amyloidoses. The focus includes tech-
niques for visualizing early steps on the amyloid formation pathway, methods for capturing
and characterizing oligomeric, potentially toxic, intermediates, strategies for preparing and
characterizing mature amyloid fi brils, and approaches for understanding templating and
transmission of amyloid aggregates. The target audience includes biochemists and bio-
physicists with an interest in elucidating the mechanisms of protein amyloid formation, as
well as chemists, pharmacologists, and clinicians with an interest in leveraging an under-
standing of such mechanisms for the purpose of therapeutic development.
Chapter 1 treats methods to prepare posttranslationally modifi ed amyloid proteins with
a focus on the production of phosphorylated forms of the Parkinson’s disease-associated
protein alpha-synuclein. Posttranslational modifi cations of synuclein and other amyloid
proteins are often associated with disease pathology yet their role in disease etiology has
remained unclear, in part because of the diffi culty of producing homogeneously modifi ed
proteins for in vitro studies. Chapter 2 describes both chemical synthesis and native chemi-
cal ligation strategies for the production of isotopically labeled amyloid proteins for charac-
terization by spectroscopic techniques such as two-dimensional infrared spectroscopy or
NMR spectroscopy. Detailed procedures are provided for the production of the diabetes-
linked amyloidogenic peptide amylin as well as for the amyloid-forming protein alpha-beta
crystallin. Chapter 3 describes the use of paramagnetic relaxation enhancement NMR spec-
troscopy to detect and describe the earliest interactions between amyloid monomers, using
alpha-synuclein as an example, while Chapter 4 describes the use of circular dichroism
spectroscopy to detect the presence of helical intermediates during the formation of amy-
loid fi brils, in this case using amylin as an example. The formation of helical intermediates
has been implicated as a potentially critical step in the formation of fi brillar aggregates by a
number of amyloid proteins. Chapter 5 describes innovative applications of fl uorescence
correlation spectroscopy to measure oligomer formation both for purifi ed amyloid proteins
in vitro and also for fl uorescently labeled amyloid proteins in intact cells, providing a unique
approach to observing the amyloid formation process in vivo using huntingtin exon 1 poly-
peptides as an example. Chapter 6 describes the application of advanced Raman Spectroscopy
methods for the characterization of the process by which amyloid fi brils form, allowing for
the characterization of different fi bril regions, such as the core or the surface, as well as for
determining the order in which secondary structure is formed during fi bril assembly. The
method is illustrated using amyloid formation by lysozyme. Chapter 7 describes an innova-
tive use of quantitative electron microscopy to determine the parameters that govern the
fi brillization kinetics of the Alzheimer’s protein tau.
Chapters 8 , 9 , and 10 focus on the characterization of oligomeric species formed on the
pathway of amyloid fi bril assembly. Chapter 8 describes the use of a powerful combination
of mass spectrometry techniques: ion mobility spectrometry and electrospray ionization, in
order to characterize the gas phase collision cross sections of different oligomeric species
v
vi Preface
that are present simultaneously in samples undergoing amyloid fi bril assembly. The
heterogeneity of species in such samples has long been a major hurdle to characterizing the
fi bril formation process, and this technique is one of very few that is able to provide a simul-
taneous analysis and resolution of different species. An application to the aggregation of
beta-2-m icroglobulin is described. Chapter 9 describes techniques to produce stable homo-
geneous preparations of oligomers formed by alpha-synuclein as well as a variety of meth-
ods to characterize these oligomers, including SDS-PAGE, circular dichroism, electron
microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and fl uores-
cence assays of phospholipid vesicle permeabilization. Chapter 10 also treats the character-
ization of oligomers formed from the protein alpha-synuclein but describes the application
of a novel single-molecule fl uorescence photobleaching approach to characterizing the
number density of the oligomers. Despite intense efforts, reliably determining the distribu-
tions of the number of molecules in amyloid oligomers has remained a frustrating chal-
lenge, and this method provides a reliable solution to this long-standing issue.
Chapters 1 1 through 1 4 describe methods for the characterization of mature amyloid
fi brils. Chapter 1 1 describes protocols for the preparation of alpha-synuclein amyloid fi brils
for characterization by solid-state NMR spectroscopy. Solid-state NMR has provided some
of our most detailed insights into the structures of amyloid fi brils of a number of proteins
and continues to be at the forefront of amyloid fi bril structure determination. Key to the
success of this method, however, is the production of high quality samples of fi brils, and
this chapter provides an avenue for achieving this. Chapter 12 describes the characteriza-
tion of amyloid fi brils formed from the protein tau using electron paramagnetic resonance
spectroscopy, which can produce information on local environments within fi brils, provide
powerful distance constraints on fi bril conformation, and also distinguish different fi bril
populations. Chapter 13 describes the preparation of amyloid fi brils for structure determi-
nation by x-ray crystallography. This approach has provided the highest resolution struc-
tural views of the central spines of a variety of amyloid-forming sequences. In addition, this
method has recently succeeded in resolving the atomic resolution structures of amyloid
oligomers, and the preparation of such samples is also described. Chapter 14 describes
methods for the analysis of amyloid fi bril structure using amide proton hydrogen exchange
monitored by NMR spectroscopy. This approach can provide information, at the single
residue level, on solvent accessible regions and hydrogen bonding patterns within fi brils.
Chapters 15 , 16 , and 17 describe computational approaches towards understanding
the structure and assembly of amyloid aggregates. This is a rapidly growing area that pro-
vides novel insights that are diffi cult or impossible to obtain via experimental methods.
Chapter 15 describes a protocol for executing replica exchange molecular dynamics simula-
tions of amyloid proteins, using a fragment of the protein tau as an example. Chapter 16
describes the use of molecular dynamics simulations to model the structures of amyloid ion
channels, as well as to calculate their ion permeation properties, using the Alzheimer’s
amyloid-beta (A-beta) peptide as an example. Ion channel formation by amyloid oligomers
is an important potential mechanism for the toxic effects of such species. Chapter 17
describes a protocol for a method that employs Bayesian statistics to leverage experimental
data on amyloid proteins for the identifi cation of ensembles of model structures that “best”
represent the experimental observables, including statistical parameters to evaluate the
signifi cance of various properties of the resulting ensembles.
Chapters 1 8 , 1 9 , and 2 0 describe methods for evaluating processes that may infl uence
the toxicity and pathology of amyloid proteins in vivo. Chapter 18 describes experimental
methods for evaluating the ability of amyloid species to permeabilize phospholipid bilayers
Preface vii
using amylin as an example. Indiscriminant membrane permeabilization by amyloid species
has been proposed as a potentially general mechanism for their toxicity. Chapter 1 9 describes
protocols to study the transmission of amyloid species between cells, using the example of
alpha-synuclein. Cell-to-cell spread of amyloid species, potentially via a prion-like mecha-
nism, has emerged as an area of tremendous interest and may explain observations of how
amyloid pathology spreads through the body and brain in neurodegenerative disease.
Chapter 20 describes the preparation of amyloid fi brils seeded using material obtained from
diseased human or mouse brain tissues in a way that preserves the ultrastructure of the
material in the original tissues, using Alzheimer’s brain derived A-beta fi brils as an example.
Such samples can then be characterized structurally, in this example using solid-state NMR,
in order to delineate the structural basis for different disease-associated fi brillar states and
to characterize amyloid strains. The possibility that different amyloid strains, corresponding
to different molecular structures of amyloid fi brils, may be associated with different disease
presentation and phenotype goes hand-in-hand with the idea that a single or a small num-
ber of nucleating aggregation events in the brain or body can lead, via cell-to-cell transmis-
sion, to a single or a few dominant fi bril forms.
In summary, this volume presents modern methods and protocols for characterizing
amyloid aggregation, amyloid aggregates, and amyloid spread and toxicity from the very
earliest manifestations in the form of nucleating conformers or transiently interacting
monomers, through the formation of helical intermediates, oligomeric species, membrane-
bound oligomers or channels, and fi nally arriving at mature amyloid fi brils, which can be
spread from cell to cell, and the molecular details of which may underlie the specifi c features
of human disease presentation.
New York, NY, USA D avid Eliezer

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
PART I LABELING STRATEGIES
1 Semisynthesis and Enzymatic Preparation of Post-translationally
Modified α-Synuclein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bruno F auvet and H ilal A . Lashuel
2 I sotope-Labeled Amyloids via Synthesis, Expression, and Chemical
Ligation for Use in FTIR, 2D IR, and NMR Studies. . . . . . . . . . . . . . . . . . . . 21
Tianqi O . Zhang , M aksim Grechko , Sean D. M oran ,
and M artin T. Z anni
PART II KINETICS/MECHANISM
3 Intermolecular Paramagnetic Relaxation Enhancement (PRE) Studies
of Transient Complexes in Intrinsically Disordered Proteins . . . . . . . . . . . . . . 45
Maria K . J anowska and Jean B aum
4 D etection of Helical Intermediates During Amyloid Formation
by Intrinsically Disordered Polypeptides and Proteins . . . . . . . . . . . . . . . . . . . 55
Andisheh Abedini , P ing Cao , and Daniel P . R aleigh
5 F luorescence Correlation Spectroscopy: A Tool to Study Protein
Oligomerization and Aggregation In Vitro and In Vivo. . . . . . . . . . . . . . . . . . 67
Bankanidhi Sahoo , K enneth W . D rombosky , and Ronald Wetzel
6 D eep UV Resonance Raman Spectroscopy for Characterizing
Amyloid Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Joseph D. Handen and Igor K . L ednev
7 Analyzing Tau Aggregation with Electron Microscopy . . . . . . . . . . . . . . . . . . 1 01
Carol J . Huseby and Jeff K uret
PART III OLIGOMERS
8 Characterization of Amyloid Oligomers by Electrospray Ionization-Ion
Mobility Spectrometry-Mass Spectrometry (ESI-IMS-MS) . . . . . . . . . . . . . . . 115
Charlotte A . Scarff , Alison E . A shcroft , and Sheena E . Radford
9 F ormation and Characterization of α-Synuclein Oligomers . . . . . . . . . . . . . . . 1 33
Wojciech Paslawski , N ikolai Lorenzen , and Daniel E. Otzen
10 F luorescence Methods for Unraveling Oligomeric Amyloid Intermediates. . . . 1 51
Niels Zijlstra , Nathalie S childerink , and V inod Subramaniam
ix