Oxidative Folding of Peptides and Proteins: Volume 15 (RSC Biomolecular Sciences) - Hardcover

Hardcover

ISBN 10: 0854041486 ISBN 13: 9780854041480

Publisher: Royal Society of Chemistry, 2008

Synopsis

With contributions from experts in the field, this book provides a comprehensive overview of the oxidative folding of cysteine-rich peptides.

About this title

About the Author

Johannes Buchner is based in the Department of Chemistry at the Technical University of Munich. His research interests focus on protein folding in vivo and in vitro, molecular chaperones, folding catalysts, antibody production and engineering. He is author and co-author of 150 publications including the Protein Folding Handbook. Luis Moroder is based at the Max Planck Institute. His research interests focus on biorganic chemistry, peptide chemistry, structure-function studies of bioactive peptides, conformational analysis and protein engineering. He is author and co-author of over 500 publications.

From the Back Cover

The formation of disulfide bonds is probably the most influential modification of peptides and proteins. An elaborate set of cellular machinery exists to catalyze and guide this process. In recent years, significant developments have been made in both our understanding of the in vivo situation and the in vitro manipulation of disulfide bonds. This is the first monograph to provide a comprehensive overview of this exciting and rapidly developing area. It offers in-depth insights into the mechanisms of in vivo and in vitro oxidative folding of proteins as well as mono- and multiple-stranded peptides. Procedures applied for laboratory and industrial purposes are also discussed by top experts in the field. The book describes the enzymes involved in the correct oxidative folding of cysteine-containing proteins in prokaryotes and eukaryotes. It then goes on to discuss the mimicking of these enzymes for successful in vitro folding of proteins (including synthetic replicates) and to deal with important issues concerning cysteine-rich peptides. The ability of natural bioactive peptides to fold correctly, and in high yields, to form defined structural motifs using cysteine sequence patterns is still puzzling. With this in mind, synthetic procedures for establishing native cysteine frameworks are discussed using selected examples, such as the potential of selenocysteines. The biotechnological and pharmaceutical relevance of proteins, peptides, their variants and synthetic replicates is continuously increasing. Consequently, this book is invaluable for peptide and protein chemists involved in related research and production.

Excerpt. © Reprinted by permission. All rights reserved.

Oxidative Folding of Peptides and Proteins

By Johannes Buchner, Luis Moroder

The Royal Society of Chemistry

Copyright © 2009 Royal Society of Chemistry
All rights reserved.

ISBN: 978-0-85404-148-0

Contents

Chapter 1 Oxidative Folding of Proteins in vivo, 
Chapter 2 Oxidative Folding of Proteins in vitro, 
Chapter 3 Redox Potentials of Cysteine Residues in Peptides and Proteins: Methods for their Determination Dallas L. Rabenstein, 
Chapter 4 Engineered Disulfide Bonds for Protein Design Luis Moroder, Hans-Jürgen Musiol and Christian Renner, 
Chapter 5 Selenocysteine as a Probe of Oxidative Protein Folding Joris Beld, Kenneth J. Woycechowsky and Donald Hilvert, 
Chapter 6 Oxidative Folding of Peptides in vitro, 
Chapter 7 Cystine-based Scaffolds for Functional Miniature Proteins Rudolf K Allemann, 
Chapter 8 Selenocystine Peptides – Synthesis, Folding and Applications Markus Muttenthaler and Paul F. Alewood, 
Subject Index, 419, 

CHAPTER 1

Oxidative Folding of Proteins in vivo

CHAPTER 1.1

Thioredoxins and the Regulation of Redox Conditions in Prokaryotes

CARSTEN BERNDT AND ARNE HOLMGREN

The Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden

The cellular redox state is a crucial mediator of several aspects of life, e.g. growth and apoptosis. It is based on low-molecular-weight thiols such as glutathione (GSH) and protein thiols, providing a reducing or an oxidizing environment. The cytoplasm with its low redox potential favors the reduction of cysteinyl residues, whereas the prokaryotic periplasm supports disulfide-bond formation. In principle thiol-disulfide pairs in proteins have two possible functions. First, disulfides often can contribute to the overall structure and stability of the protein; second, the redox state of the cysteinyl residues can regulate the activity of the protein. The modifications of cysteine residues and thereby the redox state of the particular compartment are controlled by thiol-disulfide oxidoreductases, which mainly belong to the thioredoxin family of proteins.

1.1.1 The Thioredoxin Family of Proteins

Escherichia coli thioredoxin 1 (Trx1), the first member of the thioredoxin family of proteins, was discovered more than 40 years ago as an electron donor for ribonucleotide reductase (RNR). In all organisms, this enzyme is essential for DNA synthesis during both replication and repair. The second member of the Trx family, glutaredoxin (Grx), was discovered as a GSH-dependent electron donor for RNR in an E. coli mutant lacking Trx. Today, the Trx protein family is first and foremost defined by a structural motif named the Trx fold. In spite of considerable variation in overall structure, the Trx fold is present in a variety of functionally different proteins: thiol-disulfide oxidoreductases, disulfide isomerases, glutathione S-transferases, thiol-dependent peroxidases and chloride intracellular channels.

1.1.1.1 The Thioredoxin Fold

The Trx fold motif consists of a central four-stranded β-sheet surrounded by three α-helices (Figure 1.1.1A). This basic βαβα ββα topology can only be found in bacterial glutaredoxins, while thioredoxins contain an additional β-sheet and α-helix at the N-terminus (Figure 1.1.1B,C). Variations of this motif have also been identified in domains of 723 proteins, e.g. the Trx fold domains of E. coli DsbA, a protein necessary for disulfide-bond formation in the periplasm15 (Figure 1.1.1D).

Hallmarks of the Trx motif are a cis-proline residue located before β-sheet three and the Cys-X-X-Cys active site motif located on the loop connecting β-sheet one and α-helix one. The nature and composition of these two amino acids dramatically affects the standard redox potential of the particular proteins. In E. coli, the strongest reductant, cytosolic Trx (Cys-Gly-Pro-Cys), has a redox potential of ΔE'0 = — 270 mV, the strongest oxidant, DsbA (Cys-Pro-His-Cys), has a redox potential of ΔE'0 = — 122 mV. Mutation of the Cys-Gly-Pro-Cys active site in Trx to the corresponding Cys-Pro-His-Cys active site of DsbA resulted in an increase of its standard midpoint potential to ΔE'0 = — 204 mV. Supportingly, the redox potential of a DsbA mutant harboring the Cys-Gly-Pro-Cys active site of Trx decreased by 92 mV. Several other amino acids outside the active site motif have been determined as important for the redox potential of Trx fold oxidoreductases.

1.1.1.2 Thioredoxins and the Thioredoxin System

In 1968 E. coli Trxl was sequenced, revealing the characteristic Cys-Gly-Pro-Cys active site motif2 and seven years later the Trx fold was described for the first time. Since then, more than 200 structures of different Trxs were solved including structures of both oxidized and reduced Trxs. These structures are very similar, but reduction induces local conformational changes in the area of the active site (e.g. ref. 23). The two cysteinyl residues in the active site of Trx are utilized to reduce the protein disulfide formed during the catalytic cycle of RNR.' Today we know Trx as a general disulfide reductase reducing disulfide bonds by a ping-pong mechanism (Figure 1.1.2). The N-terminal active site thiol of Trxs possesses an unusual low pKa value, whereas the pKa of the C-terminal active site is higher than that of cysteine in solution. The low pKa of the N-terminal cysteine of the E. coli Trxl active site was shown to be related to the carboxyl group of Asp 26 and the ε-amino group of Lys 57. Hence, the thiol group of the N-terminal active site cysteine is readily deprotonated under physiological conditions. Recently, it was shown by single molecule force-clamp spectroscopy that efficient catalysis requires a reorientation of the substrate disulfide bond. This investigation demonstrated that the rate-limiting step of Trx activity is the orientation of the N-terminal active site cysteine of Trx and the two disulfide bridged cysteines of the substrate in a 180° angle. This reorientation provides the condition for the nucleophilic attack of the N-terminal Cys resulting in a covalent intermediate mixed disulfide between the Trx N-terminal active site and one of the substrate's cysteinyl side-chains. The C-terminal active site cysteinyl side-chain reduces this disulfide yielding the reduced substrate and a disulfide in the active site of Trx. Subsequently, the disulfide in the active site of Trx is reduced by the dimeric flavo-enzyme thioredoxin reductase (TrxR) at the expense of NADPH (for a more detailed overview, see ref. 12).

E. coli contains two thioredoxins. Trxl, a protein with a molecular mass of 12 kD, and Trx2, a protein of 15.5 kDa, which contains an N-terminal domain of 32 amino acids including two additional Cys-X-X-Cys motifs. These four extra cysteines are able to coordinate zinc.

1.1.1.3 Glutaredoxins and the Glutaredoxin System

Glutaredoxins (Grxs) exist in all glutathione (GSH)-containing life forms. As described in Section 1.1.1.1 bacterial Grxs displayed the basic architecture of the Trx fold. Similar to Trxs, the structural comparison of reduced and oxidized E. coli Grx1 revealed more or less identical overall structures but significant changes in and around the active site.

Based on their active site motifs, Grxs can be divided into two major categories: the dithiol Grxs (active site: Cys-Pro-Tyr-Cys) and the monothiol Grxs (active site: Cys-Gly-Phe-Ser). Dithiol Grxs are general thiol-disulfide oxidoreductases reducing some protein disulfides like that in E. coli ribonucleotide reductase with a dithiol mechanism as described for Trxs above (Figure 1.1.2). In addition, Grxs are able to reduce protein-GSH mixed disulfides (de-glutathionylation) utilizing a mechanism that requires only the N-terminal active site residue (monothiol mechanism; for a more detailed overview, see ref. 33). Grxs use GSH as electron donor. The resulting glutathione disulfide (GSSG) is reduced by glutathione reductase (GR) with electrons from NADPH' (Figure 1.1.2). The molecular mechanism and the functions of monothiol glutaredoxins, which are prevalent inactive in (dithiol) Grx-specific activity assays, are only beginning to emerge (for a recent review see ref. 36).

E. coli contains the three dithiol Grxs 1-3, and the monothiol Grx4.Grxs 1, 3 and 4 are single domain proteins with molecular masses of 10, 9 and 13 kDa, respectively, whereas Grx2 is a larger protein of 24kDa. Only the N-terminal part of Grx2 is a Trx-fold domain; the overall structure resembles that of GSH-S- transferases. Recently, several monothiol Grxs including E. coli Grx4 were described as [FeS] proteins. The cofactors are coordinated by the N-terminal active site cysteine of two Grx monomers and two non-covalently bound molecules of GSH as described before for human Grx2 and poplar GrxCl. So far, E. coli Grx4 is the only essential member of the Trx family of proteins in E. coli.

1.1.1.4 NrdH and Other Related Proteins

E. coli NrdH (9 kDa) shows similarities to Grxs in its secondary and tertiary structure. However, it lacks the GSH binding site and is therefore not reduced by GSH. In vitro and in vivo studies identified TrxR as an electron donor of NrdH. NrdH was shown to act as electron donor for RNRs.

YbbN/Trxsc is a protein of 31 kDa expressed in the cytoplasm E. coli. Its N-terminus is homologous to Trxs, but without a Cys-X-X-Cys motif.

Peroxiredoxins (reviewed in ref. 49), a ubiquitous family of thiol-dependent proteins reducing peroxides like hydrogen peroxide or peroxinitrite, contain Trx-fold domains. In E. coli, four of these peroxiredoxins are present. The primary scavenger of endogenous hydrogen peroxide is alkyl hydroperoxide peroxidase C (AhpC), which is reduced by AhpF. Thiol peroxidase (Tpx), the gene product of btuE, a GSH peroxidase homolog, and bacterioferritin comi-gratory protein (BCP) are reduced by Trxl, or at least are Trx dependent.

GSH-S-transferases, as described for E. coli Grx2, contain a Trx domain. The E. coli genome encodes for eight of these proteins transferring GSH to electrophilic compounds and thereby detoxifying them.

In the periplasm, oxidative folding takes place performed by proteins of the Dsb family (reviewed in ref. 54 and Chapter 1.2). DsbA introduces disulfides into substrates, and the protein disulfide isomerases DsbC and DsbG reduce incorrect formed disulfide bonds. All these proteins are Trx-fold oxido-reductases. The eukaryotic counterparts to E. coli Dsbc/DsbG, for instance human and yeast PDIs (protein disulfide isomerases), are Trx-fold proteins as well.

1.1.2 Functions of Thioredoxin and Glutaredoxin

As in the chapters above we will focus on E. coli as the best investigated prokaryotic organism. Here, numerous functions have been described for Trxs and Grxs, both as electron donors as well as regulators of cellular function in response to oxidative stress (Figure 1.1.3).

Within the framework of this chapter, it is important to note that a number of functions described for these proteins are independent from their oxidoreductase activity, for instance E. coli Trx as subunit of the T7 DNA polymerase complex, or its activity as molecular (co-)chaperone (ref. 60, see 1.1.3.).

1.1.2.1 Regulation of Redox Conditions

Trxs and Grxs keep a reduced environment in the cytoplasm by reducing protein disulfides and protein-GSH mixed disulfides.

By modulating the redox state of protein thiols Trxs and Grxs can function as regulators of transcription factors. OxyR is such a factor activating transcription of several E. coli genes encoding proteins defending against hydrogen peroxide induced oxidative stress, e.g. catalase 1, AhpCF, GSH reductase, Grx1 and Trx2. The formed disulfide, which activates OxyR, is most likely in vivo reduced by Grx1. The activity of the transcription factor SoxR is regulated by a [2Fe2S] cluster. If the cluster is oxidized, the transcription of proteins involved in defense against oxidative stress is activated. In vitro, the reconstitution of the [FeS] cluster of SoxR is promoted by the Trx system. In addition, SoxR inactivation is inhibited in E. coli mutants lacking TrxR and GR.

Trx and Grx have also been described as electron donors for antioxidant enzymes, for instance, peroxiredoxins as described in Section 1.1.1.4.

Methionine sulfoxides that may form during oxidative stress by reactive oxygen species are reduced by methionine sulfoxide reductases (Msrs). In E. coli six Msrs are present, which are most likely using Trxl as the electron donor in vivo.66 Since increased expression of Grxl and Trx2 in an E. coli mutant lacking Trxl allows growth using methionine sulfoxide as the sole source of methionine, these two oxidoreductants are also potential electron donors for Msrs in vivo.

1.1.2.2 Regulation of Metabolic Enzymes

Trx and Grx were first described as electron donors for RNR. RNRs catalyze the reduction of nucleotides to deoxynucleotides and are thus essential for DNA synthesis. E. coli cells are equipped with three RNR enzymes. While RNR1a and RNRIII are essential for aerobic and anaerobic metabolism, respectively, RNR1b is not essential. The rate-limiting step during enzymatic turn-over of ribonucleotides by RNR la is the reduction of a disulfide performed by Trxs or Grxs. In E. coli, Grx1 may be the primary electron donor for RNR1a in vivo. Trxl may be involved in activation of RNRIII via reduction of a disulfide.

Bacteria are able to satisfy their need for reduced sulfur by assimilation of inorganic sulfate. Reduction of sulfate (SO2-4) to sulfide (S2-) requires eight electrons and takes place in two steps. First, sulfate is activated to adeny-lylsulfate (APS) by ATP sulfurylase and then to phoshoadenylylsulfate (PAPS) by APS kinase and subsequently reduced to sulfite (SO2-3) by PAPS reductase. Secondly, sulfite is reduced to sulfide using six electrons provided by NADPH (reviewed in ref. 139). Trxl and Grxl were identified as electron donors for APS kinase and PAPS reductase in E. coli.

Arsenate reductase (ArsC) detoxifies arsenate to arsenite with electrons provided by E. coli Grxs 1, 2 and 3. The substrate for Grxs is a glutathio-nylated arsenate intermediate.

[FeS] clusters, found in all life forms, can undergo reversible redox reactions, determine protein structure, act as catalytic centers and as sensitive sensors of iron and various oxygen species. The biosynthesis of these cofactors is therefore essential for catalytic function of several enzymes. During biosynthesis sulfide and iron are delivered to a scaffold protein, which coordinates the newly synthesized [FeS] cluster before transferring it to target proteins. In E. coli, two independent systems for [FeS] cluster biosynthesis are present: the Isc (iron sulfur cluster) and the Suf (mobilization of sulfur) systems. The Trx system was shown to mediate iron binding of IscA and SufA, proteins that have been described as alternative scaffold proteins, or as potential iron donors for the formation of [FeS] clusters in the scaffold IscU. Monothiol Grxs including E. coli Grx4 are crucially involved in iron-sulfur cluster biosynthesis and regulation of iron homeostasis.

1.1.3 Thioredoxins, Glutaredoxins and Protein Folding

Trxs are by far the most well known members of the Trx family. The investigations on these proteins, as well as Grxs, provided general insights in functions and mechanisms of all members of the Trx family. Even though most of these initial general experiments were done in vitro, they provided important concepts for the in vivo situation. The different ways in which Trxs and Grxs can participate in prokaryotic but also eukaryotic protein folding are the subject of this chapter.

1.1.3.1 Regulation of Protein Folding via Electrons Provided by Thioredoxins and Glutaredoxins

Trxs and Grxs has been described as regulators of many proteins involved in folding via their redox activities.

In prokaryotes, Trx and Grx catalyze the reduction of protein disulfides in E. coli Hsp33. This reduction led to the formation of Zn coordinating inactive monomers. Under conditions of oxidative stress, Hsp33 forms dimers protecting unfolded proteins against aggregation (see also Chapter 1.8).

Yeast Ssp2 and human Hdj2, two redox-regulated co-chaperones, are reduced by Trxs.

As described in Section 1.1.1.4 peroxiredoxins interact with the Trx system. Some eukaryotic peroxiredoxins were characterized as chaperones. H2O2-induced chaperone activity of yeast and human Prxs 2 requires the active site cysteinyl side-chains and the Trx system as cofactor. In concert with yeast Prx2, the Trx system protects ribosomes against stress-induced aggregation.

Electrons provided by Trxl are also essential for the correct oxidative folding of proteins in the E. coli periplasm (Figure 1.1.4, black), where the two protein disulfide isomerases DsbC and DsbG reduce incorrect formed disulfide bonds for further isomerization. A constant electron supply is guaranteed by the membrane protein DsbD, which itself is reduced by cytoplasmic Trx.

Several proteomic approaches identified molecular chaperones as new targets of the Trx and Grx systems: human Trxl was identified as interaction partner of 14-3-3 ζ proteins during interphase and mitosis in HeLa cells. Synergistically with α-crystallin, the human Trx system can recover inactivated GR in human aged clear and cataract lenses. Cyclophilin, a peptidyl-prolyl cis-trans isomerase essential for protein folding is activated via reduction by spinach Trx-m. Cyclophilin was also found in investigations aiming at the identification of new targets of A. thaliana cytosolic Trx-h3 and spinach plastidic Trx-m. Using poplar Grx-C4 as bait Rouhier et al. identified a 14-3-3 protein (At5g6543), Hsp60, Hsp70 and cyclophilin as interacting proteins in extracts of different plants. The following human chaperones were identified as targets for reversible glutathionylation:

Hsp10, Hsp60, Hsp70, Hsp90, Hsc70, 14-3-3 proteins, cyclophilin A, and Cox17. As mentioned in Section 1.1.1.3 Grxs are highly specific for protein-GSH mixed disulfides, which suggests that all the above-listed chaperone-GSH mixed disulfides are likely targets for Grxs in vivo. Indeed, it was shown that glutathionylated human Hsc70 is a substrate for Grx1, and that reduction of the mixed disulfide decreased chaperone activity.

(Continues...)

Excerpted from Oxidative Folding of Peptides and Proteins by Johannes Buchner, Luis Moroder. Copyright © 2009 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2008
• Language: English
• ISBN 10: 0854041486
• ISBN 13: 9780854041480
• Binding: Hardcover
• Number of pages: 448
• Editor: Luis Moroder