Oxidative Folding of Proteins

Basic Principles, Cellular Regulation and Engineering

Matthias J Feige

• Publisher: Royal Society of Chemistry
• Year: 2018
• eBook ISBN: 9781788014854
• Edition: 1
• Topic: Ciencias biológicas
• Subtopic: Bioquímica

The formation of disulphide bonds is probably the most influential modification of proteins. These bonds are unique among post-translational modifications of proteins as they can covalently link cysteine residues far apart in the primary sequence of a protein. This has the potential to convey stability to otherwise marginally stable structures of proteins. However, the reactivity of cysteines comes at a price: the potential to form incorrect disulphide bonds, interfere with folding, or even cause aggregation. An elaborate set of cellular machinery exists to catalyze and guide this process: facilitating bond formation, inhibiting unwanted pairings and scrutinizing the outcomes. Only in recent years has it become clear how intimately connected this cellular machinery is with protein folding helpers, organellar redox balance and cellular homeostasis as a whole.

This book comprehensively covers the basic principles of disulphide bond formation in proteins and describes the enzymes involved in the correct oxidative folding of cysteine-containing proteins. The biotechnological and pharmaceutical relevance of proteins, their variants and synthetic replicates is continuously increasing. Consequently this book is an invaluable resource for protein chemists involved in realted research and production.

CHAPTER 1.1

Disulfide Bonds in Protein Folding and Stability

Matthias J. Feige^*a, Ineke Braakman^*b and Linda M. Hendershot^*c

^aDepartment of Chemistry and Institute for Advanced Study, Technische Universität München, 85748 Garching, Germany

^bCellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, The Netherlands

^cDepartment of Tumor Cell Biology, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
^*Email: [email protected]; [email protected]; [email protected]

Disulfide bonds are unique among post-translational modifications, as they add covalent crosslinks to the polypeptide chain. Accordingly, they can exert pronounced effects on protein folding and stability. This is of particular importance for secreted or cell surface proteins, where disulfide bonds are abundant and serve to stabilize proteins against unfolding and dissociation in the extracellular milieu. However, in addition to these bonds providing security to a natively folded protein or aiding the folding process by stabilizing folding intermediates, the cysteines that form these bonds can be perilous during the maturation of nascent polypeptide chains as they enter the endoplasmic reticulum where the concentration of unfolded proteins approaches millimolar levels. This danger is even greater if the native bonds ultimately form between non-consecutive cysteines that are distant in the linear sequence or if non-native bonds are a prerequisite to achieving the final, functional structure of a protein. A wealth of exquisite detail has been obtained from in vitro studies on the biophysical effects of disulfide bonds on protein folding. Correspondingly, in-depth in vivo studies have established that the same principles apply to oxidative folding in a cell, but reveal a much more complex folding trajectory for many of the proteins that have been examined. In this chapter, we review the biophysical properties of disulfide bonds and how they affect the structure and folding of individual proteins. Based on this, we discuss similarities and differences between in vitro and in vivo folding reactions. The types of disulfide bonds that form during co-translational protein folding are described, as are the cellular strategies for accommodating this risk-laden covalent modification. We conclude with a discussion of the impact of disulfide bonds on protein misfolding and human disease.

1.1.1Stabilization of Proteins by Disulfide Bonds

Early protein folding studies were interpreted as suggesting that the native state of a protein corresponds to one well-defined conformation, whereas the unfolded state corresponds to a random coil.¹ If no other states than either the native or unfolded state are kinetically or thermodynamically stable, we speak of a two-state folding mechanism for a protein.² This scenario set the stage for early analyses of the role that disulfide bonds play in protein stability.

An ideal random coil is devoid of any long-range interactions except excluded volume effects. It behaves as a freely joined chain with segments of defined length.³ In such a system, the impact of a covalent crosslink between two defined residues of the polypeptide chain, such as a disulfide bond, would be greatest on the unfolded state, significantly decreasing the conformational freedom of the random coil. Restricting the conformational space of the unfolded state reduces its entropy. Hence, in the presence of a disulfide bond, the entropy change for the reaction to the ordered native state is less negative, with net stabilization of the folded protein as a result. This model will be called the chain-entropy model in the following sections. A quantitative description was developed by Flory,⁴ Schellman⁵ and Poland and Scheraga.⁶ The decrease in entropy of the unfolded state is derived from the probability that two otherwise free elements of the chain are now found in a defined volume element (v). The mathematical description of the problem, based on polymer theory, can be found in the equation

(1.1.1)

where R is the gas constant and l the average length of a statistical segment of the chain composed of N segments; in proteins, l is assumed to be 3.8 Å, corresponding to one amino acid. A major point of discussion has been the suitable choice of v. A value of 57.9 ų based on the closest possible approach of two thiols is mostly in use.⁷ Hence eqn (1.1.1) can be simplified to

(1.1.2)

where n is the number of amino acids bridged by the disulfide bond. Based on a study of ribonuclease (RNase) T₁ with no, one and two intact disulfide bonds, eqn (1.1.2) was developed by Pace et al.⁷ They not only found a good correlation between n and ΔΔG upon removal of disulfide bonds in RNase T₁, but also observed agreement between the predictions from these equations and the experimental data for lysozyme, RNase A and the antibody C_L domain.

The above equations have two main consequences. Conceptually, the stabilization of a protein is thought to be an entirely entropy-driven process with an impact exclusively on the unfolded state. In theory, the stabilization achieved by a disulfide bond should therefore always increase with increase in the number of amino acids between the two cysteines. Despite its appealing simplicity, in practice this theory falls short in important aspects of real proteins. It treats the unfolded polypeptide chain as a system devoid of any intra- or intermolecular interactions. Furthermore, the water surrounding a protein is an important factor in shaping the free energy landscape of the polypeptide chain, and also in the native state, but is neglected in the equations.^8–10 The impact of these considerations regarding disulfide bonds was addressed by Doig and Williams in 1991.¹¹ They argued that disulfide bonds may significantly decrease the solvent-accessible surface in the unfolded state of a protein. As a consequence, hydrophobic residues and also hydrogen-bond donors and acceptors may become buried. Burial of hydrophobic residues would lead to less ordering of water and thus a higher entropy of the solvent surrounding disulfide-containing proteins. Consequently, the hydrophobic effect, a major driving force in protein folding, should be less pronounced. On the other hand, hydrogen bonding with the solvent will be less extensive for a more compact unfolded state, thus reducing this competition. As a result, folding to the native state will be enthalpically more favorable. According to the authors, this enthalpic contribution must be considered as the major stabilizing factor of disulfide bonds. This model will therefore be called the solvent-enthalpy model in the following sections. As in the chain-entropy model, effects on the native state are also neglected in this model. Both models are summarized in Figure 1.1.1.

Figure 1.1.1 Models for the role of disulfide bonds on polypeptide stability. (A) The chain-entropy model predicts a smaller change in entropy (ΔS) upon folding of a polypeptide chain containing a disulfide bond than of one lacking a disulfide bond. This leads to a net stabilization of the native state. (B) The solvent-enthalpy model predicts fewer solvent–polypeptide interactions (water molecules are displayed in a CPK representation and hydrogen bonds as dashed lines) and less exposure of hydrophobic residues for a polypeptide chain containing a disulfide bond than for a polypeptide chain lacking one. This is assumed to reduce the enthalpy change (ΔH) upon loss of solvent–polypeptide interactions during folding and thus will lead to net stabilization of the native state.

A variety of experimental evidence argues either for or against these two theories, rendering the problem much more complex, but the data obtained from the various experiments also provide a chance for further insights. The advent of site-specific mutagenesis offe...

Table of contents

• Cover
• Title
• Copyright
• Contents
• Section I: Principles and Analysis of Disulfide Bond Formation
• Chapter 1.1 Disulfide Bonds in Protein Folding and Stability 3
• Chapter 1.2 Techniques to Monitor Disulfide Bond Formation and the Reduction Potential of Cysteine–Cystine Couples In vitro and In vivo34
• Chapter 1.3 Real-time Detection of Thiol Chemistry in Single Proteins
• Chapter 1.4 Analysis of Disulfide Bond Formation in Therapeutic Proteins 81
• Section II: Disulfide Bonds in Peptides and Proteins: Structure, Function and Evolution
• Chapter 2.1 Evolutionary Adaptations to Cysteine-rich Peptide Folding 101
• Chapter 2.2 In vitro Refolding of Proteins 129
• Chapter 2.3 Allosteric Disulfide Bonds 152
• Section III: Oxidative Folding in the Cell
• Chapter 3.1 Disulfide Bond Formation and Isomerization in Escherichia coli177
• Chapter 3.2 Disulfide Bond Formation in Mitochondria 205
• Chapter 3.3 Structural Insights into Disulfide Bond Formation and Protein Quality Control in the Mammalian Endoplasmic Reticulum 224
• Chapter 3.4 Mechanisms of Oxidative Protein Folding and Thiol-dependent Quality Control: Tales of Cysteines and Cystines 249
• Chapter 3.5 Disulfide Bond Formation Downstream of the Endoplasmic Reticulum 267
• Section IV: Oxidative Folding and Cellular/Organism Homeostasis
• Chapter 4.1 How Microbes Cope with Oxidative Stress 287
• Chapter 4.2 Disulfide Bond Formation in the Endoplasmic Reticulum 306
• Chapter 4.3 Redox Regulation of Hsp70 Chaperone Function in the Endoplasmic Reticulum 334
• Chapter 4.4 Thioredoxin and Cellular Redox Systems: Beyond Protein Disulfide Bond Reduction 355
• Section V: Engineering Covalent Linkages in Peptides and Proteins
• Chapter 5.1 Stabilization of Peptides and Proteins by Engineered Disulfide Bonds 381
• Chapter 5.2 Genetic Code Expansion Approaches to Introduce Artificial Covalent Bonds into Proteins In Vivo 399
• Subject Index