Oxidative folding of proteins: basic principles, cellular regulation and engineering

450 Pages·2018·29.521 MB·English

by  Feige, Matthias Johannes

Oxidative Folding of Proteins
Basic Principles, Cellular Regulation and Engineering
Chemical Biology
Editor-in-chief:
Tom Brown, University of Oxford, UK
Series editors:
Kira J. Weissman, Lorraine University, France
Sabine Flitsch, University of Manchester, UK
Nick J. Westwood, University of St Andrews, UK
Titles in the series:
1: High Throughput Screening Methods: Evolution and Refinement
2: Chemical Biology of Glycoproteins
3: Computational Tools for Chemical Biology
4: Mass Spectrometry in Chemical Biology: Evolving Applications
5: Mechanisms of Primary Energy Transduction in Biology
6: Cyclic Peptides: From Bioorganic Synthesis to Applications
7: DNA-targeting Molecules as Therapeutic Agents
8: Protein Crystallography: Challenges and Practical Solutions
9: Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and
Engineering
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Oxidative Folding of Proteins
Basic Principles, Cellular Regulation and
Engineering
Edited by
Matthias J. Feige
TU München, Germany
Email: [email protected]
Chemical Biology No. 9
Print ISBN: 978-1-78262-990-0
PDF ISBN: 978-1-78801-325-3
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Foreword
Throughout the 50 year history of the protein folding field, the tracking of 
disulfide bonds and their formation has played a central role. In pioneering 
days when folding studies were limited to refolding of denatured proteins 
in vitro, the making, breaking and rearrangement of disulfide bonds pro-
vided the only reliable way to monitor kinetics and conformations during 
the refolding process. Later, when it became possible to study folding in live 
cells, the stepwise generation of intramolecular disulfides in nascent and 
newly synthesized proteins was what we and others followed. The assembly 
of oligomeric proteins could also be easily detected if they acquired inter-
chain disulfides. Misfolding could often be identified by the formation of 
disulfide-bonded aggregates. For larger proteins, such as influenza virus 
hemagglutinin, it was possible to characterize both co-translational and 
post-translational folding events simply by following mobility differences of 
folding intermediates in nascent and fully synthesized polypeptide chains 
using non-reducing SDS-PAGE.
By combining such “disulfide tracking” approaches with radioactive pulse 
and chase, sufficient time resolution was achieved to follow the entire folding 
process, especially of larger multi-domain proteins. When combined with 
conformation-specific antibodies, inhibitors, mutations, limited proteolysis, 
expression of recombinant proteins and other perturbations, this approach 
revealed insights into the rules that govern the folding program of many pro-
teins. The role of chaperones and folding enzymes and the influence of covalent 
modifications such as glycosylation could be analyzed.
The reviews in this volume show that great progress has been made in 
understanding protein maturation, quality control, secretion and endoplas-
mic reticulum (ER)-associated degradation. A deeper understanding of the 
compartments where disulfide bonds are formed is emerging. We have, 

Chemical Biology No. 9
Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering
Edited by Matthias J. Feige
© The Royal Society of Chemistry 2018
Published by the Royal Society of Chemistry, www.rsc.org
v
vi Foreword
for example, learned that the ER lumen provides an exquisitely complex, adapt-
able environment for the oxidative folding of protein products produced  
by a cell. It contains a powerful mixture of folding factors that together support 
a continuous process of folding, unfolding, misfolding and refolding. For 
most newly synthesized proteins, this is accompanied by oxidation, reduction 
and rearrangement of disulfides. The mixture of chaperones, thiol oxidoreduc-
tases, folding sensors, ER-associated degradation components, modifying 
enzymes, Ca2+ buffers, membrane transporters and other critical factors are 
fine tuned according to cell function and physiological condition.
It is important to realize that our understanding of in vivo folding is still 
superficial. The cell biology of oxidative protein folding still presents us with 
formidable challenges. Folding factors work together via complicated syner-
gies in a highly complex, dynamic milieu that we cannot successfully mimic 
in vitro. In the ER, we do not know the concentrations of chaperones or their 
distribution within the luminal space. We do not comprehend their mutual 
interactions and influences and we cannot predict the choice and combina-
tion of factors needed for specific protein substrates. We hardly have a clue 
about the physical state of the interior of the ER. Is it a fluid or gel like? We 
do not know how selective transport of cargo from the ER is maintained and 
how quality control really works. A connection between defects in protein 
folding and ER function is evident in various disease states, but we cannot 
address such problems effectively. Hence there is still a lot to accomplish in 
this important field of research.
Ari Helenius
Institute of Biochemistry
ETH Zürich
Zürich, Switzerland
Preface
Most naturally evolved proteins are only marginally stable, always on the verge 
of losing their structure. But this is what most exciting biology is about: flex-
ible molecular machines that undergo large conformational changes in addi-
tion to small fluctuations and adjustments, together conferring functionality. 
Nature has developed a means to reconcile these controversies: the disulfide 
bond. Strategically placed, this covalent crosslink of two cysteine residues can 
drive folding, inhibit unfolding and stabilize oligomeric protein assemblies. 
But it comes with a cost: owing to the intrinsic reactivity of cysteines, disulfide 
bonds may form erroneously, may need to be isomerized or broken for a pro-
tein to fold correctly, or for a protein to be degraded. Furthermore, reactions 
that form or break disulfide bonds are redox reactions – which means that the 
cell has to handle potentially harmful oxidative species.
Despite these odds, the intricate machinery that cells have developed to 
make, break and isomerize disulfide bonds speaks to the importance of this 
covalent modification in proteins. Disulfide bonds are particularly abun-
dant in extracellular proteins, where they confer stability to the structure 
of individual polypeptide chains and their oligomeric assemblies. As such, 
unpaired cysteines are an important feature recognized by the secretory pro-
tein quality control machinery as a signature of incomplete protein folding 
and assembly.
However, beyond these structural roles, it is becoming increasingly evi-
dent that cysteines and disulfide bonds can do more: they serve regulatory 
roles, can stabilize protein conformations with different functions, and sig-
nal changes in the redox environment of a protein. The intimate connection 
between cysteines, disulfide bonds and protein folding and function also 
explains their prominent role in human disease, where cysteine mutations are 
common.

Chemical Biology No. 9
Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering
Edited by Matthias J. Feige
© The Royal Society of Chemistry 2018
Published by the Royal Society of Chemistry, www.rsc.org
vii
viii Preface
Apart from these basic biological considerations, the rise of recombinant 
proteins and biopharmaceuticals depends on proper ways to form disulfide 
bonds – and techniques to analyze them. But it also depends on approaches 
to introduce covalent modifications into proteins for the purpose of their 
stabilization without disrupting functionality.
Both the basis of and recent developments in our understanding of 
these various facets of disulfide bonds are covered in this volume by lead-
ing experts in their field. The 18 individual contributions are combined in 
five sections: the first section is dedicated to the principles and analysis of 
disulfide bond formation – from single molecules to biopharmaceuticals. 
The second section covers structural and functional roles of disulfide bonds, 
from evolutionary, biotechnological and mechanistic perspectives. The third 
section traces the question of how disulfide bonds form, are isomerized and 
are broken – with a view to revealing common principles between differ-
ent kingdoms of life and between different mammalian organelles. Based 
on these insights, the fourth section addresses the question of how oxida-
tive folding and cellular homeostasis are coupled and how cells avoid and 
deal with oxidative stress. Lastly, the fifth section provides insights into how 
covalent linkages in proteins can be engineered for structural and functional 
purposes.
Covering such a broad scope of topics depends on the commitment of 
many people: my gratitude goes to all the authors of chapters in this volume 
and also to Rowan Frame, Drew Gwilliams and Robin Driscoll at the RSC, 
who made it possible to develop this comprehensive and in-depth overview 
of the principles, the biology, the analysis and the design of oxidative protein 
folding.
Matthias J. Feige
Department of Chemistry and Institute for Advanced Study
Technische Universität München
Garching, Germany
Contents
Section I: Principles and Analysis of Disulfide Bond
Formation
Chapter 1.1 Disulfide Bonds in Protein Folding and Stability 3
Matthias J. Feige, Ineke Braakman and
Linda M. Hendershot
  1.1.1  Stabilization of Proteins by Disulfide  
Bonds   3
  1.1.2  Disulfide Bonds in Protein Folding Reactions: 
Biophysical Considerations   8
  1.1.3  Distinctions Between In vitro Refolding  
Assays and Protein Biosynthesis in a Cell   11
  1.1.4  Disulfide Bonds in ER Protein Folding   14
  1.1.5  Formation of Disulfide Bonds Between  
Sequential Cysteines   16
  1.1.6  Disulfide Bonds Between Non-sequential,  
Often Long-range, Cysteines   17
  1.1.7  Non-native Disulfide Bonds as a Prerequisite  
to Correct Protein Maturation   19
  1.1.8  Disulfide Bonds, Protein Misfolding and  
Human Disease   20
  1.1.9  Concluding Thoughts   24
   Acknowledgements   25
   References   25

Chemical Biology No. 9
Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering
Edited by Matthias J. Feige
© The Royal Society of Chemistry 2018
Published by the Royal Society of Chemistry, www.rsc.org
ix