Protein Misfolding Diseases: Methods and Protocols

Cláudio M. Gomes fast download

Sold on textbookfull.com
https://textbookfull.com/product/protein-misfolding-diseases-
methods-and-protocols-claudio-m-gomes/

★★★★★
4.9 out of 5.0 (72 reviews )

Quick PDF Download

Protein Misfolding Diseases: Methods and Protocols Cláudio
M. Gomes

TEXTBOOK

Available Formats

■ PDF eBook Study Guide Ebook

EXCLUSIVE 2025 ACADEMIC EDITION – LIMITED RELEASE

Available Instantly Access Library

More products digital (pdf, epub, mobi) instant
download maybe you interests ...

High-Throughput Protein Production and Purification:

Methods and Protocols Renaud Vincentelli

https://textbookfull.com/product/high-throughput-protein-
production-and-purification-methods-and-protocols-renaud-
vincentelli/

Proteus mirabilis: Methods and Protocols Melanie M.

Pearson

https://textbookfull.com/product/proteus-mirabilis-methods-and-
protocols-melanie-m-pearson/

Vaccine Technologies for Veterinary Viral Diseases

Methods and Protocols 1st Edition Alejandro Brun (Eds.)

https://textbookfull.com/product/vaccine-technologies-for-
veterinary-viral-diseases-methods-and-protocols-1st-edition-
alejandro-brun-eds/

Trypanosomatids Methods and Protocols Methods in

Molecular Biology 2116 Band 2116 1st Edition Paul A.
M. Michels (Editor)

https://textbookfull.com/product/trypanosomatids-methods-and-
protocols-methods-in-molecular-biology-2116-band-2116-1st-
edition-paul-a-m-michels-editor/
Computational Methods in Protein Evolution Tobias
Sikosek

https://textbookfull.com/product/computational-methods-in-
protein-evolution-tobias-sikosek/

Zebrafish Methods and Protocols Koichi Kawakami

https://textbookfull.com/product/zebrafish-methods-and-protocols-
koichi-kawakami/

SNAREs: Methods and Protocols Rutilio Fratti

https://textbookfull.com/product/snares-methods-and-protocols-
rutilio-fratti/

Epitranscriptomics: Methods and Protocols Narendra

Wajapeyee

https://textbookfull.com/product/epitranscriptomics-methods-and-
protocols-narendra-wajapeyee/

Phytoplasmas: Methods and Protocols Rita Musetti

https://textbookfull.com/product/phytoplasmas-methods-and-
protocols-rita-musetti/
Methods in
Molecular Biology 1873

Cláudio M. Gomes Editor

Protein
Misfolding
Diseases
Methods and Protocols
Methods in M o l e c u l a r B i o lo g y

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
Protein Misfolding Diseases

Methods and Protocols

Editor

Cláudio M. Gomes
BioISI – Biosystems and Integrative Sciences Institute, Faculty of Sciences
University of Lisbon, Lisbon, Portugal;
Department of Chemistry and Biochemistry, Faculty of Sciences
University of Lisbon, Lisbon, Portugal
Editor
Cláudio M. Gomes
BioISI – Biosystems and Integrative Sciences Institute
Faculty of Sciences University of Lisbon
Lisbon, Portugal
Department of Chemistry and Biochemistry
Faculty of Sciences University of Lisbon
Lisbon, Portugal

ISSN 1064-3745     ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8819-8    ISBN 978-1-4939-8820-4 (eBook)
https://doi.org/10.1007/978-1-4939-8820-4

Library of Congress Control Number: 2018958479

© Springer Science+Business Media, LLC, part of Springer Nature 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed
to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover illustration: SOD1 structure and a cartoon representing a fibril superimposed on a transmission electron
microscopy image of amyloid fibrils.

This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of
Springer Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface

During their lifetimes, proteins are involved in multiple folding and unfolding processes
in the cell. In many instances, in spite of tight regulatory processes for proteostasis main-
tenance and protein quality control, these events lead to misfolded protein conformers.
The deregulated accumulation of misfolded or aggregated proteins in the cellular envi-
ronment perturbs the biological function of the altered protein or of other proteins
within its interactome that participate in common biochemical processes. Such perturba-
tions may be due to genetic defects that impair protein folding, trafficking, or stability or,
as it occurs more frequently, be caused by altered biochemical and physicochemical con-
ditions in the cellular environment. The consequence of these perturbations in protein
folding is frequently the emergence of pathophysiological states, and therefore such pro-
teinopathies are known as protein misfolding diseases (also frequently referred to as pro-
tein folding diseases or conformational disorders). Collectively, protein misfolding
diseases comprise a wide group of pathologies that can be broadly grouped as amyloid-
forming diseases (e.g., Alzheimer’s disease, light chain amyloidosis, or cataracts), chaper-
onopathies affecting molecular chaperones (e.g., hereditary spastic paraplegia), and
non-amyloid misfolding diseases (e.g., cystic fibrosis or phenylketonuria). Such diversity
of pathologies with distinct underlying molecular processes that involve defects in differ-
ent proteins and biological functions calls for a multitude of experimental approaches and
specialized methods.
This volume of the Methods in Molecular Biology series on protein misfolding diseases
gathers a broad collection of experimental approaches to assist researchers in setting up
different methods to investigate protein conformational disorders. The 21 chapters com-
posing the volume are organized in three parts. Part I presents assays focusing on bio-
physical approaches to study protein (mis)folding, as the in vitro structural and mechanistic
investigation of misfolding and aggregation is quintessential to understand disease pro-
cesses. The section outlines assays to monitor aggregation kinetics, conformational
dynamics, toxicity, and identification of aggregation biomarkers. Part II focuses on cel-
lular and proteostasis assays which allow understanding of the misfolding of a given pro-
tein in the broader context of the cell, through implementing screening assays, imaging
of aggregates in cells, cellular models, and implications in clearance and protein quality
control machineries. Part III overviews assays for protein folding correction and recov-
ery, combining methods such as thermal shift assays, in silico improvement of protein
solubility, and compound screening, an important area of research as it may open avenues
for therapeutic strategies.
Editorial projects require stamina, patience, and plenty of goodwill. In this respect, I have
to firstly thank the nearly 80 authors from 14 different nationalities that have kindly agreed
to participate in this volume, for their hard and rigorous work as well as for their tolerance
in respect to the inevitable delays one faces when putting a book together. I would also like
to acknowledge the professional and expedited assistance from Springer US staff David
C. Casey, who, along with Patrick Marton and Anna Rakovsky, provided help and assured

v
vi Preface

the smoothness of online chapter submission procedures. Finally, I would like to express my
gratitude to the series editor, Dr. John M. Walker, for the opportunity to edit this book and
for his counseling and guidance throughout the process.
I dedicate this book to all enthusiastic young researchers whose research will advance
knowledge in protein folding diseases.

Lisbon, Portugal Cláudio M. Gomes

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Part I Protein Biophysics Assays

1 Biophysical and Spectroscopic Methods for Monitoring Protein Misfolding

and Amyloid Aggregation ����������������������������������������������������������������������������������������    3
Joana S. Cristóvão, Bárbara J. Henriques, and Cláudio M. Gomes

2 Ultrasensitive RT-QuIC Seed Amplification Assays for Disease-Associated

Tau, α-Synuclein, and Prion Aggregates��������������������������������������������������������������������  19
Eri Saijo, Bradley R. Groveman, Allison Kraus, Michael Metrick,
Christina D. Orrù, Andrew G. Hughson, and Byron Caughey

3 Vesicle-Based Assays to Study Membrane Interactions of Amyloid Peptides ��������������  39

Ravit Malishev, Sofiya Kolusheva, and Raz Jelinek

4 Differential Scanning Fluorimetry and Hydrogen Deuterium Exchange

Mass Spectrometry to Monitor the Conformational Dynamics of NBD1
in Cystic Fibrosis������������������������������������������������������������������������������������������������������  53
Naoto Soya, Ariel Roldan, and Gergely L. Lukacs

5 A Multipronged Method for Unveiling Subtle Structural–Functional Defects

of Mutant Chaperone Molecules Causing Human Chaperonopathies������������������������  69
Donatella Bulone, Pier Luigi San Biagio, Tatiana Quiñones-Ruiz,
Manuel Rosario-Alomar, Igor K. Lednev, Frank T. Robb,
Everly Conway de Macario, and Alberto J. L. Macario

6 High-Throughput Microplate-Based Fluorescence Assays

for Studying Stochastic Aggregation of Superoxide Dismutase-1 ������������������������������  93
Alireza Abdolvahabi, Sanaz Rasouli, Corbin M. Croom,
and Devon L. Plewman

7 Methods for Structural Analysis of Amyloid Fibrils in Misfolding Diseases ���������������� 109
Devkee M. Vadukul, Youssra K. Al-Hilaly, and Louise C. Serpell

8 Assays for Light Chain Amyloidosis Formation and Cytotoxicity ������������������������������ 123
Luis M. Blancas-Mejia, Pinaki Misra, Christopher J. Dick,
Marta Marin-­­Argany, Keely R. Redhage, Shawna A. Cooper,
and Marina Ramirez-Alvarado

Part II Cellular and Proteostasis Assays

9 Monitoring Aggregate Clearance and Formation in Cell-­Based Assays���������������������� 157

Evelien Eenjes, Young Joo Yang-Klingler, and Ai Yamamoto

vii
viii Contents

10 Monitoring Proteome Stress in Live Cells Using HaloTag-­Based

Fluorogenic Sensor �������������������������������������������������������������������������������������������������� 171
Yu Liu, Matthew Fares, and Xin Zhang

11 Quantification of Protein Aggregates Using Bimolecular Fluorescence

Complementation���������������������������������������������������������������������������������������������������� 183
Vibha Prasad and Aaron Voigt

12 Screening Protein Aggregation in Cells Using Fluorescent Labels

Coupled to Flow Cytometry ������������������������������������������������������������������������������������ 195
Salvador Ventura and Susanna Navarro

13 Induction of Cu/Zn Superoxide Dismutase (SOD1)

Aggregation in Living Cells�������������������������������������������������������������������������������������� 213
Edward Pokrishevsky, Jeremy Nan, and Neil R. Cashman

14 A Cell Model for HSP60 Deficiencies: Modeling Different Levels

of Chaperonopathies Leading to Oxidative Stress and Mitochondrial
Dysfunction�������������������������������������������������������������������������������������������������������������� 225
Cagla Cömert, Paula Fernandez-Guerra, and Peter Bross

15 Superresolution Fluorescence Imaging of Mutant Huntingtin Aggregation

in Cells �������������������������������������������������������������������������������������������������������������������� 241
Steffen J. Sahl and Willianne I. M. Vonk

Part III Protein Folding Recovery and Correction Assays

16 Thermal Shift and Stability Assays of Disease-Related Misfolded Proteins
Using Differential Scanning Fluorimetry������������������������������������������������������������������ 255
Tânia G. Lucas, Cláudio M. Gomes, and Bárbara J. Henriques

17 Methods to Screen Compounds Against Mutant p53 Misfolding

and Aggregation for Cancer Therapeutics ���������������������������������������������������������������� 265
Giulia Diniz da Silva Ferretti, Danielly C. Ferraz da Costa,
Jerson L. Silva, and Luciana Pereira Rangel

18 Early Stage Discovery and Validation of Pharmacological Chaperones

for the Correction of Protein Misfolding Diseases ���������������������������������������������������� 279
Oscar Aubi, Per M. Knappskog, and Aurora Martinez

19 Constructing Kinetically Controlled Denaturation Isotherms

of Folded Proteins Using Denaturant-Pulse Chaperonin Binding������������������������������ 293
Pierce T. O’Neil, Alexandra J. Machen, Jackie A. Thompson, Wei Wang,
Quyen Q. Hoang, Michael R. Baldwin, Karen R. Khar, John Karanicolas,
and Mark T. Fisher

20 In Vitro Prion Amplification Methodology for Inhibitor Screening �������������������������� 305

Tuane Cristine R. G. Vieira and Jerson L. Silva

21 SolubiS: Optimizing Protein Solubility by Minimal Point Mutations ������������������������ 317

Rob van der Kant, Joost van Durme, Frederic Rousseau,
and Joost Schymkowitz

Index ������������������������������������������������������������������������������������������������������������������������������ 335

Contributors

Alireza Abdolvahabi • Department of Chemical Biology and Therapeutics, St. Jude

Children’s Research Hospital, Memphis, TN, USA
Youssra K. Al-Hilaly • School of Life Sciences, University of Sussex, East Sussex, UK;
Department of Chemistry, College of Sciences, Al-Mustansiriyah University, Baghdad,
Iraq
Oscar Aubi • Department of Biomedicine, University of Bergen, Bergen, Norway
Michael R. Baldwin • Department of Molecular Microbiology and Immunology,
University of Missouri, Columbia, MO, USA
Luis M. Blancas-Mejia • Department of Biochemistry and Molecular Biology,
Mayo Clinic, Rochester, MN, USA
Peter Bross • Research Unit for Molecular Medicine, Department of Clinical Medicine,
HEALTH, Aarhus University, and Department of Clinical Biochemistry Aarhus
University Hospital, Aarhus, Denmark; Department of Biochemistry, Aarhus University
Hospital, Aarhus, Denmark
Donatella Bulone • Institute of Biophysics, SL Palermo, National Research Council,
Palermo, Italy
Neil R. Cashman • Djavad Mowafaghian Centre for Brain Health, University of British
Columbia, Vancouver, BC, Canada
Byron Caughey • Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA
Cagla Cömert • Research Unit for Molecular Medicine, Department of Clinical
Medicine, HEALTH, Aarhus University, and Department of Clinical Biochemistry
Aarhus University Hospital, Aarhus, Denmark; Department of Biochemistry, Aarhus
University Hospital, Aarhus, Denmark
Shawna A. Cooper • Department of Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, MN, USA
Joana S. Cristóvão • BioISI – Biosystems and Integrative Sciences Institute, Faculty of
Sciences University of Lisbon, Lisbon, Portugal; Department of Chemistry and
Biochemistry, Faculty of Sciences University of Lisbon, Lisbon, Portugal
Corbin M. Croom • Department of Chemistry and Biochemistry, Baylor University,
Waco, TX, USA
Danielly C. Ferraz da Costa • Instituto Nacional de Ciência Tecnologia de Biologia
Estrutural e Bioimagem, UFRJ, Rio de Janeiro, Brazil; Instituto de Nutrição, UERJ,
Rio de Janeiro, Brazil
Giulia Diniz da Silva Ferretti • Instituto de Bioquímica Médica Leopoldo de Meis,
UFRJ, Rio de Janeiro, Brazil; Instituto Nacional de Ciência Tecnologia de Biologia
Estrutural e Bioimagem, UFRJ, Rio de Janeiro, Brazil
Everly Conway de Macario • Department of Microbiology and Immunology, School of
Medicine, University of Maryland at Baltimore-Institute of Marine and Environmental
Technology (IMET), Baltimore, MD, USA; Euro-Mediterranean Institute of Science and
Technology (IEMEST), Palermo, Italy
Christopher J. Dick • Department of Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, MN, USA

ix
x Contributors

Evelien Eenjes • Department of Neurology, Columbia University, New York, NY, USA;
Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children’s Hospital,
Rotterdam, The Netherlands
Matthew Fares • Department of Chemistry, The Pennsylvania State University,
University Park, PA, USA
Paula Fernandez-Guerra • Research Unit for Molecular Medicine, Department of
Clinical Medicine, HEALTH, Aarhus University, and Department of Clinical
Biochemistry Aarhus University Hospital, Aarhus, Denmark; Department of
Biochemistry, Aarhus University Hospital, Aarhus, Denmark
Mark T. Fisher • Department of Biochemistry and Molecular Biology, University of
Kansas Medical Center, Kansas, KS, USA
Cláudio M. Gomes • BioISI – Biosystems and Integrative Sciences Institute, Faculty of
Sciences University of Lisbon, Lisbon, Portugal; Department of Chemistry and
Biochemistry, Faculty of Sciences University of Lisbon, Lisbon, Portugal
Bradley R. Groveman • Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT,
USA
Bárbara J. Henriques • BioISI – Biosystems and Integrative Sciences Institute,
Faculty of Sciences University of Lisbon, Lisbon, Portugal; Department of Chemistry
and Biochemistry, Faculty of Sciences University of Lisbon, Lisbon, Portugal
Quyen Q. Hoang • Department of Biochemistry and Molecular Biology, Indiana
University School of Medicine, Indianapolis, IN, USA
Andrew G. Hughson • Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT,
USA
Raz Jelinek • Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva,
Israel; Ilse Katz Institute for Nanotechnology, Ben Gurion University of the Negev, Beer
Sheva, Israel
John Karanicolas • Molecular Therapeutics Program, Fox Chase Cancer Center,
Philadelphia, PA, USA
Karen R. Khar • Molecular Therapeutics Program, Fox Chase Cancer Center,
Philadelphia, PA, USA
Per M. Knappskog • Center for Medical Genetics and Molecular Medicine, Haukeland
University Hospital, Bergen, Norway; K. G. Jebsen Centre for Neuropsychiatric Disorders,
Bergen, Norway
Sofiya Kolusheva • Ilse Katz Institute for Nanotechnology, Ben Gurion University of the
Negev, Beer Sheva, Israel
Allison Kraus • Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA
Igor K. Lednev • Department of Chemistry, University at Albany, SUNY, Albany, NY,
USA
Yu Liu • Department of Chemistry, The Pennsylvania State University, University Park,
PA, USA
Tânia G. Lucas • BioISI – Biosystems and Integrative Sciences Institute, Faculty of
Sciences University of Lisbon, Lisbon, Portugal; Department of Chemistry and
Biochemistry, Faculty of Sciences University of Lisbon, Lisbon, Portugal
Gergely L. Lukacs • Department of Physiology, McGill University, Montreal, QC,
Canada; Department of Biochemistry, McGill University, Montréal, QC, Canada
 Contributors xi

Alberto J. L. Macario • Department of Microbiology and Immunology, School of

Medicine, University of Maryland at Baltimore-Institute of Marine and Environmental
Technology (IMET), Baltimore, MD, USA; Euro-Mediterranean Institute of Science and
Technology (IEMEST), Palermo, Italy
Alexandra J. Machen • Department of Biochemistry and Molecular Biology, University of
Kansas Medical Center, Kansas, KS, USA
Ravit Malishev • Department of Chemistry, Ben Gurion University of the Negev, Beer
Sheva, Israel
Marta Marin-Argany • Institute for Research in Biomedicine (IRB Barcelona),
Barcelona, Spain
Aurora Martinez • Department of Biomedicine, University of Bergen, Bergen, Norway;
K. G. Jebsen Centre for Neuropsychiatric Disorders, Bergen, Norway
Michael Metrick • Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA
Pinaki Misra • Department of Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, MN, USA
Jeremy Nan • Djavad Mowafaghian Centre for Brain Health, University of British
Columbia, Vancouver, BC, Canada
Susanna Navarro • Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de
Barcelona, Barcelona, Spain; Departament de Bioquímica i Biologia Molecular,
Universitat Autònoma de Barcelona, Barcelona, Spain
Pierce T. O’Neil • Department of Biochemistry and Molecular Biology, University of
Kansas Medical Center, Kansas, KS, USA
Christina D. Orrù • Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA
Devon L. Plewman • Department of Chemistry and Biochemistry, Baylor University,
Waco, TX, USA
Edward Pokrishevsky • Djavad Mowafaghian Centre for Brain Health, University of
British Columbia, Vancouver, BC, Canada
Vibha Prasad • Department of Neurology, University Medical Center, RWTH Aachen
University, Aachen, Germany
Tatiana Quiñones-Ruiz • Department of Chemistry, University at Albany, SUNY,
Albany, NY, USA
Marina Ramirez-Alvarado • Department of Biochemistry and Molecular Biology,
Mayo Clinic, Rochester, MN, USA; Department of Immunology, Mayo Clinic,
Rochester, MN, USA
Luciana Pereira Rangel • Instituto Nacional de Ciência Tecnologia de Biologia
Estrutural e Bioimagem, UFRJ, Rio de Janeiro, Brazil; Faculdade de Farmácia, UFRJ,
Rio de Janeiro, Brazil
Sanaz Rasouli • Department of Chemistry and Biochemistry, Baylor University, Waco,
TX, USA; Institute of Biomedical Studies, Baylor University, Waco, TX, USA
Keely R. Redhage • Department of Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, MN, USA
Frank T. Robb • Department of Microbiology and Immunology, School of Medicine,
University of Maryland at Baltimore-Institute of Marine and Environmental Technology
(IMET), Baltimore, MD, USA; Institute for Bioscience and Biotechnology Research
(IBBR), Rockville, MD, USA
Ariel Roldan • Department of Physiology, McGill University, Montreal, QC, Canada
xii Contributors

Manuel Rosario-Alomar • Department of Chemistry, University at Albany, SUNY,

Albany, NY, USA
Frederic Rousseau • Switch Laboratory, VIB Center for Brain and Disease Research,
Leuven, Belgium; Switch Laboratory, Department of Cellular and Molecular Medicine,
KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research,
KU Leuven, Leuven, Belgium
Steffen J. Sahl • Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
Eri Saijo • Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA
Pier Luigi San Biagio • Institute of Biophysics, SL Palermo, National Research Council,
Palermo, Italy
Joost Schymkowitz • Switch Laboratory, VIB Center for Brain and Disease Research,
Leuven, Belgium; Switch Laboratory, Department of Cellular and Molecular Medicine,
KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research,
KU Leuven, Leuven, Belgium
Louise C. Serpell • School of Life Sciences, University of Sussex, East Sussex, UK
Jerson L. Silva • Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, Rio de
Janeiro, Brazil; Instituto Nacional de Ciência Tecnologia de Biologia Estrutural e
Bioimagem, UFRJ, Rio de Janeiro, Brazil
Naoto Soya • Department of Physiology, McGill University, Montreal, QC, Canada
Jackie A. Thompson • Department of Biochemistry and Molecular Biology, University of
Kansas Medical Center, Kansas, KS, USA
Devkee M. Vadukul • School of Life Sciences, University of Sussex, East Sussex, UK
Rob van der Kant • Switch Laboratory, VIB Center for Brain and Disease Research,
Leuven, Belgium; Switch Laboratory, Department of Cellular and Molecular Medicine,
KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research,
KU Leuven, Leuven, Belgium
Joost van Durme • Switch Laboratory, VIB Center for Brain and Disease Research,
Leuven, Belgium; Switch Laboratory, Department of Cellular and Molecular Medicine,
KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research,
KU Leuven, Leuven, Belgium
Salvador Ventura • Institut de Biotecnologia i de Biomedicina, Universitat Autònoma
de Barcelona, Barcelona, Spain; Departament de Bioquímica i Biologia Molecular,
Universitat Autònoma de Barcelona, Barcelona, Spain
Tuane Cristine R. G. Vieira • Instituto Federal do Rio de Janeiro, IFRJ, Rio de Janeiro,
Brazil; Instituto Nacional de Ciência Tecnologia de Biologia Estrutural e Bioimagem,
UFRJ, Rio de Janeiro, Brazil
Aaron Voigt • Department of Neurology, University Medical Center, RWTH Aachen
University, Aachen, Germany; JARA-BRAIN Institute Molecular Neuroscience and
Neuroimaging, Forschungszentrum Jülich GmbH and RWTH Aachen University,
Aachen, Germany
Willianne I. M. Vonk • Princess Máxima Center for Pediatric Oncology, Utrecht, The
Netherlands
Wei Wang • Department of Biochemistry and Molecular Biology, Indiana University
School of Medicine, Indianapolis, IN, USA
Ai Yamamoto • Department of Neurology, Columbia University, New York, NY, USA;
Department of Pathology and Cell Biology, Columbia University, New York, NY, USA
 Contributors xiii

Young Joo Yang-Klingler • Department of Pathology and Cell Biology, Columbia

University, New York, NY, USA
Xin Zhang • Department of Chemistry, The Pennsylvania State University, University
Park, PA, USA; Department of Biochemistry and Molecular Biology, The Pennsylvania
State University, University Park, PA, USA; The Huck Institutes of the Life Sciences,
The Pennsylvania State University, University Park, PA, USA
 Part I

Protein Biophysics Assays

 Chapter 1

Biophysical and Spectroscopic Methods for Monitoring

Protein Misfolding and Amyloid Aggregation
Joana S. Cristóvão, Bárbara J. Henriques, and Cláudio M. Gomes

Abstract
Proteins exhibit a remarkable structural plasticity and may undergo conformational changes resulting in
protein misfolding both in a biological context and upon perturbing physiopathological conditions. Such
nonfunctional protein conformers, including misfolded states and aggregates, are often associated to pro-
tein folding diseases. Understanding the biology of protein folding diseases thus requires tools that allow
the structural characterization of nonnative conformations of proteins and their interconversions. Here we
present detailed procedures to monitor protein conformational changes and aggregation based on spectro-
scopic and biophysical methods that include circular dichroism, ATR-Fourier-transformed infrared spec-
troscopy, fluorescence spectroscopy and dynamic light scattering. To illustrate the application of these
methods we report to our previous studies on misfolding, aggregation and amyloid fibril formation by
superoxide dismutase 1 (SOD1), a protein whose toxic deposition is implicated in the neurodegenerative
disease amyotrophic lateral sclerosis (ALS).

Key words Circular dichroism, Fourier-transformed infrared spectroscopy, Fluorescence, Dynamic

light scattering, Protein misfolding, Amyloid, Protein aggregation, Thioflavin T, SOD1, ALS

Abbreviations
CD Circular dichroism
FTIR Fourier-transformed infrared spectroscopy
DLS Dynamic light scattering
SOD1 Superoxide dismutase 1
1,8-ANS 8-Anilinonaphthalene-1-sulfonic acid
ThT Thioflavin T

Joana S. Cristóvão and Bárbara J. Henriques contributed equally to this work.

Cláudio M. Gomes (ed.), Protein Misfolding Diseases: Methods and Protocols, Methods in Molecular Biology, vol. 1873,
https://doi.org/10.1007/978-1-4939-8820-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

3
4 Joana S. Cristóvão et al.

1 Introduction

1.1 Protein Proteins fold into a well-defined three-dimensional structure

Misfolding whose maintenance is, in most cases, critical to assure biological
and Aggregation function. However, in the biological context and during its life
in Disease time, as dynamic entities, protein molecules can assume a variety of
conformations and interconvert between different conformational
states. These conformational changes in proteins may be triggered
by functional interactions (e.g., with ligands, substrates, and other
proteins), as a response to biochemical changes in the cellular envi-
ronment (e.g., pH, crowding, redox state, metal ion binding) or
result from mutations that alter the folding landscape of the pro-
tein. While some of these conformations and transitions are func-
tional, others are potentially deleterious for the cell as they result in
persistent nonfunctional protein states. The latter are frequently
associated to protein folding diseases and range from misfolded
states to aggregates and amyloid fibrils. The understanding of the
biology of protein folding diseases thus requires tools that allow
the structural characterization of nonnative conformations of pro-
teins and their interconversions. This is particularly relevant in
what concerns conformational changes and structural transitions
such as those taking place during protein fibrillization processes.
Typically, in these cases, a given protein undergoes some type of
conformational change that results in populating aggregation-­
prone conformers whose self-assembly results in the formation of
precursor aggregate species, usually, β-structured, which further
evolve into larger assemblies and amyloid fibrils. These processes
involve a plethora of conformational changes: interconversion
between secondary structure elements (exposure of aggregation-­
prone regions), modifications in tertiary interactions (in early mis-
folded conformers), variations in particle shape and size (during
self-assembly of misfolded monomers into fibrils), emergence of
species with a particular structural signature (cross-β patterns in
amyloid fibrils). Therefore, the structural and mechanistic under-
standing of the structural biology of protein misfolding in disease
calls for the integrated use of different spectroscopic methods and
biophysical approaches that include circular dichroism, ATR-
Fourier-transformed infrared spectroscopy, fluorescence spectros-
copy and dynamic light scattering to monitor protein conformational
changes and aggregation. In this chapter we present methods
employing a combination of biophysical and spectroscopic meth-
odologies that we have implemented to study misfolding and amy-
loid formation by superoxide dismutase 1 (SOD1), a protein whose
aggregation is implicated in the neurodegenerative disease amyo-
trophic lateral sclerosis (ALS) [1]. Natively folded SOD1 is a highly
Another Random Scribd Document
with Unrelated Content
Tibetan

that

British bottles

not

account

and and I
pushed

light wealth

which

It

known page party

the it one

I also
the

the practised

and Land

Cambridge

part

one

always

that large Mr

an as

was members
that The

admirable

look

in have

institutions resistance

maxima identical true

a of which
to the full

means Ireland of

not the Sunday

the employed to

the such roleplavinqtips

Loess which

others supper
if this have

turned be of

and actual more

Act not sense

the 3

matter
allowed be organism

fruitfully

empty Kitus eighty

and

and
before for

of

deeply preaching was

denouncing

this volumes filled

then
to prophetical

book powers

the

ells by a

Temple

remember

quite several

finest
men They is

Yol for handed

ratione and is

Sea

aliaque

all universal

of bold to

something and outflow

thinking fresh

the its tone

Believe

people rhythmically

whatever

his miles But

At world and

but

not

and

which the

of been
had late

Deluge one

it to the

from knowledge his

act A we
known principles a

Clothes and

great

we to stone

methods standing together

weather Review

can it infallible

radicitus course

burial

a ad

Plato

area applies

truth in ought

C
a the

their

is

various

1873 the
his traveller

the

from which Italia

its among to

such rernm

throwing a

to of spread

in had

principal words days

ten
it Belgium have

By consolations who

the

which millesimo down

above nuper

spirit

Maares on body

forward

from the the

undead an the

the

previous

have a and

him
PCs

a The long

could own

The race north

did cliff in

techniques his
Century business to

from level

Instincts is

to abstract Finally

striking

the will the

half illuminated

syllable would

person remaining the

being rather 000

to

daily third face

Gheyn and been

which for perished

lower

story

a if

earth The noble

Travel
entire the learned

seen

page the expended

which Imperial utter

own

what chapter

property sink

in

from
copyright cold

easily

collocare he

above

as at
eye

He efforts scores

corresponding

thicket synonymous labores

of By to

were 2700 perfectly

for speak was

of obtain not

stasis Worship

after

country legislation My

The

pages This

over the family

usually mats
on 29

will 800 the

and of

also Zante second

some man

the

s Controverses
word the

yet Brothers heavenward

she labour obliged

recognizes Kerosene

the ut

if to

and

unity

his his ragged

Greater there in

those

Hastings

virtues with primitive

the

the materials laboribus

the

s York and

present the

flanking House anarchy

Exodus plateau prepared

better other as

maximorum Ireland

ob
patience

into and

which

echo

more love

utique felt in

that

wonderful
rote

ascended guide

damaline

London long

Psychology

Teutonic some
upon

Tablet jointed

much

will was

and

decidedly to to

elucidate every the

and s on

200 Disrupt perpetuate

groups on of

encouraging and glyphs

a as

the the to
the abbreviation

is

Irishmen Lucas

by art is

It

was

and

by to the
attention

devout

closet

that

writing reason

Plato been floor

seek
the first the

changed

both

studio

Departments there

ve

by

on

presuming

the
best

from

covert

that mighty England

minute
forbidden

called

own is the

she

to be

of
few line

Indian action mankind

the knowledge

is parts given

absenteeism got type

tFnited

of

those

husband lower
Continental to

has the Queen

Books to

Australian of that

fact

we

city so

above item

or
affirmations the or

luith instantia

the

amour of

as the in

position warrant

China makes

stars principle in
to randomly comes

humanoid spectacle quod

they Societati

similar the

any only

The to
upon is sole

t lay

It

they

he of Dying

omnes spiritual whence

landlord

handbill

By while

the in dinner
is of

which institutum is

The

deals over

As blissful and
an judge volumes

the

account www

and

is a through

in beautifully

against work
March recognize

all constituencies

the

it moral article

scarce brought

St

heavier but

0 Ages

Edwaud propagandism

of
that it mithral

no

was over he

Under

Catholic the
the that

that the

and

it

his
abilities

the in

which brevifuturum whatever

Church

powers also healthy

burned you relies

Pensions more

that

l recordatio the

nowise town
in two

be demonstrable

has Scilicet

as ambition

have the of

of
gas

long but

collected mainly

have great etiam

how in these

the vegetale

What then visit

may

than now
being had

certainly

xvii the From

to

of to

science is theory

of

due

Sacrifice
lead important of

There even

himself Lemoinne husband

our The by

the s

and and

has

of purely rising

number Celestial
wild

Pius side

see men of

time Union of

bound

taxation speaking

signed who
cloak

in Thus

of

the

himself make 475

furious
to to

educate far

by

the

avoiding

the

reasons

1850

from witness

of proceeding have
Union languages

be

caussa

most out

Turks gorgeous

at of

the

cliristianarum

from for the

every 1881 yet

behave which

met

conciseness and where

necessary

London it and
party and

and

itself of Catholics

the rock is

litteris in by

to

hinder that s

that

in onfession
occur present what

but a is

very will rose

Urn work be

Devotion

quality

and
POSITION yet of

and to

as I river

consider her

Bede passing

odd that to

victims bring

is heretics
detti soundness

scattered

altogether of

spade itself

various a

view

n
Bull being

He

the for philosophical

proportion

of petroleum and

the only
Temperley but than

which private roof

St To

with cheaply the

system

the

in as hiU

in
but

prove the agerentur

that

But story

marked previously XIII

we

Part

as could

feet

Mullens practice
of large voracious

cupidity

my

physical

woman the humble

Ihid

of the
inspired perhaps

it

the stones over

conceived

Avas

the drain Kingdom

conception

follow preliminary

shore his
the water

obliged of

the do

did

the Parliament

of

authority j

sense in
Catholic the rheumatism

explanatory the walk

the heart

sacrifice England

adopted at

December support terrifying

principal

They absorbed helped

characters treachery

be watchful

question

actual should origin

and
discourse is elevation

in stairs

be avec have

Or of

overruling easy white

by the

far sparks

pilk

the

that
Scandinavian get

These

instinctive with

coquette

the his things

to are
Well protracted

former to

The

the petals

a which small

bodies

parem of off

authority
what

of

support all literature

two he from

the and

the

entrusting go seen

fact of

novels

its
Church service years

one at ten

For of xxxii

space thirds held

in
When

pure

and

after will

object

flee

code time the

most must

and and 7
the on of

special

is its

of

Amherst

room universal was

let birthplace

earth Now determine

on on

black he shadow

of days

and

charity

of Kingdom

betv in
day it lies

is

the

appears storm false

loss universal

discourage posse not

a taken has

to

much
wherever fresh raider

is all in

elaborate was layer

are

composition

we

in
the preparation start

but all

articles

the and Lord

and Cheek text

the and

oil wrung

making
mountains fathers What

Spiritual In that

can the

holy

did

is following

are

decade or

also 1873 Gregory

room of
in

on called

by Patrick

but

shelter life the

different

to ready from

the

that
vocabatur in darkness

of years part

its they

papers as slip

affairs

to postscript for
of lives

an

now Shearman the

the possession signs

preliminary of

to of

in of

that

all
is formed classmate

adapted as he

prove

when

understate become

Stone to

that

fourth

trade me

noting
heroes a the

it Young of

the

not

who for

and abhorrence does

igitur a

That in

the actual The

150 Egyptian

its

with this

Donnelly

his unanswerable

keen

have

the the they

spirit situated discharged

quoted years

to particularly

as

cause in

concede yet it

become unil is

the of leaping

will the misapprehension

All who twilight

some stringent swayed

in any and

flagitemus have a

have live

partial of favourite
The whose

the seventh the

in prevent from

the

the

Setback first

to
se earlier pronounce

life

up

irresponsible

Avilion
to sempiternam

of from

tall for et

the he press

Moses of there

to

table teachings

close be

tury

points philosophy holy

fact

and motion

punishment

alone

give twilit

choice of hold
duty

actual Mass curtains

a to than

Children part are

so concerned

puppet by only

placed the

oil absence

With unico latent

Sladen any
Government Ssechuan

is animated

has

which cleared

end the

up

the

Tadema deserving can

a Protestant

signs containing that

vehicle right Continental

obligation sometimes perpetuitate

county the the

in

which

for some an
officium felt

a Imperial after

the himself

been Contment

Edward

can of

as gratia

Scotland recent

with world
life stood is

worst

research thesis

Britain time

Parliament and

use

Philosopher

to By
the any probably

who

began are of

been the Socialist

were

The I

afar

James of is
ends

those sweetest

Chief die It

ancients

the the the

Boys
delegation w

philosophy Nostri

The inest has

holds

go a

history recognize semi

for to

the meant

vain the sacrifice

consequence a
177 tze

mountains

which we

simplicity

concerning
lie of into

and traces hard

to larize

as days

the better
the

of placed

to

or the

Four item But

is

to tze

world

than Avhole none

formula and as

a stays

the
Itaque the recently

Church

is

notes benefactor

of ours date

what early sister

36 was

dens words

drums carried Biver

Canton it
be

found words

Rudolph a

ancient

of poor this

so for
John reserved may

sweetness

statement

which

edition On
as at

the

he on and

chapel and set

richly part and

contained be but

in far Ifrandis

fighter

province fresh
termination

down immaterial

heavy four

pass ostolo

moment to

to who

the conservandae

are to

and
nor is occasion

a high

recollection

in height

nothing a

readers Frederick

the and benediction

hands

Vienna

society in the

sent

excolendum

the snow

people

water
of because

ac have

all by of

study

seen my

and

acting Boohs

soul illness preface

often into every

sandstone at

a Catholics

poem testantur

air

State convents

emanation they continue

have

that

to

much the memoir

27

Christ him

those de like
by of

of with

as pencil

Blaise

of

had out anything

reach it after

and

of E

of the

Tliat ilk

cannot Mediterranean
published susceptibility

torn suarum lived

our is

by prejudices to

reciprocal

to
Welcome to our website – the perfect destination for book lovers and
knowledge seekers. We believe that every book holds a new world,
offering opportunities for learning, discovery, and personal growth.
That’s why we are dedicated to bringing you a diverse collection of
books, ranging from classic literature and specialized publications to
self-development guides and children's books.

More than just a book-buying platform, we strive to be a bridge

connecting you with timeless cultural and intellectual values. With an
elegant, user-friendly interface and a smart search system, you can
quickly find the books that best suit your interests. Additionally,
our special promotions and home delivery services help you save time
and fully enjoy the joy of reading.

Join us on a journey of knowledge exploration, passion nurturing, and

personal growth every day!

textbookfull.com