Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 143-156).

Das E. coli Expressionssystem ist das am häufigsten angewandte hinsichtlich der rekombinante Proteinexpression für strukturelle und funktionelle Analysen aufgrund der hohen erzielten Ausbeuten und der einfachen Handhabbarkeit. Allerdings ist insbesondere die Expression eukaryotischer Proteine in E. coli problematisch, z.B. wenn das Protein nicht korrekt gefaltet ist und in unlöslichen Inclusion Bodies anfällt. In manchen Fällen ist die Analyse von Deletionskonstrukten oder einzelnen Proteindomänen der Untersuchung des Vollängeproteins vorzuziehen. Dies umfasst die Herstellung eines Satzes von Expressionskonstrukten, welche charakterisiert werden müssen. In dieser Arbeit werden Methoden optimiert und evaluiert für die in vitro-Faltung von Inclusion Body-Proteinen sowie die Entwicklung einer Hochdurchsatz-Charakterisierung von Expressionskonstrukten. Die Überführung von Inclusion Body-Proteinen in den nativen Zustand beinhaltet zwei Schritte: (a) Auflösen mit einen chaotropen Reagenz oder starkem ionischen Detergenz und (b) Faltung des Proteins durch Beseitigung des Chaotrops begleitet von dem Transfer in einen geeigneten Puffer. Die Ausbeute an nativ gefaltetem Protein ist oft stark eingeschränkt aufgrund von Aggregation und Fehlfaltung; sie kann allerdings durch die Zugabe bestimmter Additive zum Faltungspuffer erhöht werden. Solche Additive müssen empirisch identifiziert werden. In dieser Arbeit wurde eine Testprozedur für Faltungsbedingungen entwickelt. Zur Reduzierung der möglichen Kombinationen der getesteten Additive wurden sowohl empirische Beobachtungen aus der Literatur als auch bekannte Eigenschaften der Additive berücksichtigt. Zur Verminderung der eingesetzten Proteinmenge und des Arbeitsaufwandes wurde der Test automatisiert und miniaturisiert mittels eines Pipettierroboters. 20 Bedingungen zum schnellen Verdünnen von denaturierten Proteinen werden hierbei getestet und zwei Bedingungen zur Faltung von Proteinen mit dem Detergenz/Cyclodextrin Protein-Faltungssystem von Rozema et al. (1996). 100 µg Protein werden pro Bedingung eingesetzt. Zusätzlich werden acht Bedingungen für die Faltung von His-Tag-Fusionsproteinen (ca. 200 µg), welche an eine Metallchelat-Matrix immobilisiert sind, getestet. Die Testprozedur wurde erfolgreich angewendet zur Faltung eines humanen Proteins, der p22 Untereinheit von Dynactin, welche in E. coli in Inclusion Bodies exprimiert wird. So wie es sich bei vielen Proteinen darstellt, war auch für p22 Dynactin kein biologischer Nachweistest vorhanden, um den Erfolg des Faltungsexperimentes zu messen. Die Löslichkeit des Proteins kann nicht als eindeutiges Kriterium dienen, da neben nativ gefaltetem Protein, lösliche fehlgefaltete Spezies und Mikroaggregate auftreten können. Diese Arbeit evaluiert Methoden zur Detektion kleiner Mengen nativen Proteins nach dem automatisierten Faltungstest. Bevor p22 Dynactin gefaltet wurde, wurden zwei Modellenzyme zur Evaluierung eingesetzt, bovine Carboanhydrase II (CAB) und Malat Dehydrogenase aus Schweineherz-Mitochondrien. Die wiedererlangte Aktivität nach der Rückfaltung wurde korreliert mit verschiedenen biophysikalischen Methoden. Bindungsstudien mit 8-Anilino-1-Naphtalenesulfonsäure ergaben keine brauchbaren Informationen bei der Rückfaltung von CAB aufgrund der zu geringen Sensitivität und da fehlgefaltete Proteine nicht eindeutig von nativem Protein unterschieden werden konnten. Tryptophan Fluoreszenzspektren der rückgefalteten CAB wurden zur Einschätzung des Erfolges der Rückfaltung angewandt. Die Verschiebung des Intensitätsmaximum zu einer niedrigeren Wellenlänge im Vergleich zum denaturiert entfalteten Protein sowie die Fluoreszenzintensität korrelierten mit der wiedererlangten enzymatischen Aktivität. Für beide Modellenzyme war analytische hydrophobe Interaktionschromatographie (HIC) brauchbar zur Identifizierung rückgefalteter Proben mit aktivem Enzym. Kompakt gefaltetes, aktives Enzym eluierte in einem distinkten Peak im abnehmenden Ammoniumsulfat-Gradienten. Das Detektionslimit für analytische HIC lag bei 5 µg. Im Falle von CAB konnte gezeigt werden, dass Tryptophan-Fluoreszenz-Spektroskopie und analytische HIC in Kombination geeignet sind um Falsch-Positive oder Falsch-Negative, welche mit einem der Monitore erhalten wurden, auszuschließen. Diese beiden Methoden waren ebenfalls geeignet zur Identifizierung der Faltungsbedingungen von p22 Dynactin. Tryptophan-Fluoreszenz-Spektroskopie kann jedoch zu Falsch-Positiven führen, da in machen Fällen Spektren von löslichen Mikroaggregaten kaum unterscheidbar sind von Spektren des nativ gefalteten Proteins. Dies zusammenfassend wurde eine schnelle und zuverlässige Testprozedur entwickelt, um Inclusion Body-Proteine einer strukturellen und funktionellen Analyse zugänglich zu machen. In einem separaten Projekt wurden 88 verschiedene E. coli-Expressionskonstrukte für 17 humane Proteindomänen, welche durch Sequenzanalyse identifiziert wurden, mit einer Hochdurchsatzreinigung und –faltungsanalytik untersucht, um für die Strukturanalyse geeignete Kandidaten zu erhalten. Nach Expression in einem Milliliter im 96er Mikrotiterplattenformat und automatisierter Proteinreinigung wurden löslich exprimierte Proteindomänen direkt analysiert mittels 1D ¹H-NMR Spektroskopie. Hierbei zeigte sich, dass insbesondere isolierte Methylgruppen-Signale unter 0.5 ppm sensitive und zuverlässige Sonden sind für gefaltetes Protein. Zusätzlich zeigte sich, dass – ähnlich zur Evaluierung des Faltungstests – analytische HIC effizient eingesetzt werden kann zur Identifizierung von Konstrukten, welche kompakt gefaltetes Protein ergeben. Sechs Konstrukte, welche zwei Domänen repräsentieren, konnten schnell als tauglich für die Strukturanalyse gefunden werden. Die Struktur einer dieser Domänen wurde kürzlich von Mitarbeitern gelöst, die andere Struktur wurde im Laufe dieses Projektes von einer anderen Gruppe veröffentlicht. / For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 µg protein is used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 µg) immobilised on metal chelate resins. The screen was successfully applied to fold a human protein, the p22 subunit of dynactin that is expressed in inclusion bodies in E. coli. For p22 dynactin – as is the case for many proteins – there was no biological assay available to assess the success of the folding screen. Protein solubility can not be used as a stringent criterion because beside natively folded protein, soluble misfolded species and microaggregates may occur. This work evaluates methods to detect small amounts of natively folded protein after automated folding screening. Before folding screening with p22 dynactin, two model enzymes, bovine carbonic anhydrase II (CAB) and pig heart mitochondrial malate dehydrogenase, were used for evaluation. Recovered activity after refolding was correlated to different biophysical methods. 8-anilino-1-naphtalenesulfonic acid binding-experiments gave no useful information when refolding CAB, due to low sensitivity and because misfolded protein could not be readily distinguished from native protein. Tryptophan fluorescence spectra of refolded CAB were used to assess the success of refolding. The shift of the intensity maximum to a shorter wavelength, compared to the denaturant unfolded protein, as well as the fluorescence intensity correlated to recovered enzymatic activity. For both model enzymes, analytical hydrophobic interaction chromatography (HIC) was useful to identify refolded samples that contain active enzyme. Compactly folded, active enzyme eluted in a distinct peak in a decreasing ammonium sulfate gradient. The detection limit of analytical HIC was approx. 5 µg. In case of CAB, tryptophan fluorescence spectroscopy and analytical HIC showed that both methods in combination can be useful to rule out false positives or false negatives obtained with one method. These two methods were also useful to identify conditions for folding of p22 dynactin. However, tryptophan fluorescence spectroscopy can lead to false positives because in some cases spectra of soluble microaggregates are not well distinguishable from spectra of natively folded protein. In summary, a fast and reliable screening procedure was developed to make inclusion body proteins accessible to structural or functional analyses. In a separate project, 88 different E. coli expression constructs for 17 human protein domains that had been identified by sequence analysis were analysed using high-throughput purification and folding analysis in order to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, solubly expressed protein domains were directly analysed using 1D ¹H-NMR spectroscopy. It was found that isolated methyl group signals below 0.5 ppm are particularly sensitive and reliable probes for folded protein. In addition – similar to the evaluation of a folding screen – analytical HIC proved to be an efficient tool for identifying constructs that yield compactly folded protein. Both methods, 1D ¹H-NMR spectroscopy and analytical HIC, provided complementary results. Six constructs, representing two domains, could be quickly identified as targets that are well suitable for structural analysis. The structure of one of these domains was solved recently by co-workers, the other structure was published by another group during this project.

Protein folding and export to the membrane are crucial events in the cell. Both processes may be initiated already at the ribosome, assisted by factors that bind to the polypeptide as it emerges from the ribosome. The signal recognition particle (SRP) scans the ribosome for nascent peptides destined for membrane insertion and targets these ribosomes to the site for translocation in the membrane. Trigger factor (TF) is a folding chaperone that interacts with nascent chains to promote their correct folding, prevent misfolding and aggregation. In this thesis, we first investigated membrane targeting and insertion of two heterologous membrane proteins in E. coli by using in vitro translation, membrane targeting and cross-linking. We found that these proteins are dependent on SRP for targeting and that they initially interact with translocon components in the same way as native nascent membrane proteins. Moreover we have characterised the SRP and TF interactions with the ribosome both with cross-linking experiments and with quantitative binding experiments. Both SRP and TF bind to ribosomal L23 close to the nascent peptide exit site where they are strategically placed for binding to the nascent polypeptide. Quantitative analysis of TF and SRP binding determined their respective KD values for binding to non translating ribosomes and reveals that they bind simultaneously to the ribosome, thus having separate binding sites on L23. Finally, binding studies on ribosome nascent chain adds clues as to how TF functions as a chaperone.

One of the most challenging tasks remaining in the field of biochemistry is the one of understanding how the information within the amino acid sequence of proteins translates into a unique structure. Solving this problem would lead to endless possibilities for application in the medical and biotechnology industry. Many decades ago scientists realized that the process that facilitates the folding of a polypeptide chain could not be random and happen by chance; there needs to be direction in the folding free energy landscape. This landscape is defined by the thermodynamic factors entropy and enthalpy. The contribution made by enthalpy i.e. the contact energies from intra- and intermolecular interactions have been extensively investigated by various mutational studies. The influence of entropy on the other hand, is less well understood. My work focuses on the effect of altering the entropic components of forming the various parts of a known protein scaffold. This is done by genetic engineering in combination with biophysical characterisation and analysis. The results show effects on protein folding rates as well as on the pathway for nucleation and emphasis the ability of the folding landscape to readjust to entropic variations. Proteins are therefore not required to fold along a unique route to their final structure but can do so in several ways. The folding pathways we observe today have hence likely evolved as an adaptation to biological demands.

Proteins are the most important molecules in the cell since they take care of most of the biological functions which resemble life. To ensure that everything is working properly the cell has a rigorous control system to monitor the proper function of its proteins and sends old or dysfunctional proteins for degradation. Unfortunately, this system sometimes fails and the once so vital proteins start to misbehave or to accumulate and in the worst case scenario these undesired processes cause the death of their host. One example is Amyotrophic Lateral Sclerosis (ALS); a progressive and always fatal neurodegenerative disorder that is proposed to derive from accumulation of aberrant proteins. Over 140 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) are linked to ALS. The key event in SOD1 associated ALS seems to be the pathological formation of toxic protein aggregates as a result of initially unfolded or partly structured SOD1-mutants. Here, we have compared the folding behaviour of a set of ALS associated SOD1 mutants. Based on our findings we propose that SOD1 mediated ALS can be triggered by a decrease in protein stability but also by mutations which reduce the net charge of the protein. Both findings are in good agreement with the hypothesis for protein aggregation. SOD1 has also been found to be able to interact with mitochondrial membranes and SOD1 inclusions have been detected in the inter-membrane space of mitochondria originating from the spinal cord. The obvious question then arose; does the misfolding and aggregation of SOD1 involve erroneous interactions with membranes? Here, we could show that there is an electrostatically driven interaction between the reduced apo SOD1 protein including ALS associated SOD1-mutants and charged lipid membrane surfaces. This association process changes the secondary structures of these mutants in a way quite different from the situation found in membrane free aqueous environment. However, the result show that mutants interact with charged lipid vesicles to lesser extent than wildtype SOD1. This opposes the correlation between decreased SOD1 stability and disease progression. We therefore suggest that the observed interaction is not a primary cause in the ALS mechanism.

Enzymes are extraordinary molecules that can accelerate chemical reactions by several orders of magnitude. With recent advancements in structural biology together with classical enzymology the mechanism of many enzymes has become understood at the molecular level. During the last ten years significant efforts have been invested to understand the structure and dynamics of the actual catalyst (i. e. the enzyme). There has been a tremendous development in NMR spectroscopy (both hardware and pulse programs) that have enabled detailed studies of protein dynamics. In many cases there exists a strong coupling between enzyme dynamics and function. Here I have studied the conformational dynamics and thermodynamics of three model systems: adenylate kinase (Adk), Peroxiredoxin Q (PrxQ) and the structural protein S16. By developing a novel chemical shift-based method we show that Adk binds its two substrates AMP and ATP with an extraordinarily dynamic mechanism. For both substrate-saturated states the nucleotide-binding subdomains exchange between open and closed states, with the populations of these states being approximately equal. This finding contrasts with the traditional view of enzyme-substrate complexes as static low entropy states. We are also able to show that the individual subdomains in Adk fold and unfold in a non-cooperative manner. This finding is relevant from a functional perspective, since it allows a change in hydrogen bonding pattern upon substrate-binding without provoking global unfolding of the entire enzyme (as would be expected from a two-state folding mechanism). We also studied the structure and dynamics of the plant enzyme PrxQ in both reduced and oxidized states. Experimentally validated structural models were generated for both oxidation states. The reduced state displays unprecedented μs-ms conformational dynamics and we propose that this dynamics reflects local and functional unfolding of an α-helix in the active site. Finally, we solved the structure of S16 from Aquifex aeolicus and propose a model suggesting a link between thermostability and structure for a mesophilic and hyperthermophilic protein pair. A connection between the increased thermostability in the thermophilic S16 and residual structure in its unfolded state was discovered, persistent at high denaturant concentrations, thereby affecting the difference in heat capacity difference between the folded and unfolded state. In summary, we have contributed to the understanding of protein dynamics and to the coupling between dynamics and catalytic activity in enzymes.

Im Zebrafisch (Danio rerio) ist ein Protein identifiziert worden, das eine wichtige Rolle in der Entwicklung der Brustflossen zu besitzen scheint und als Fibin, dem englischen Akronym für Fin bud initiation factor (Flossenknospeninitiationsfaktor), bezeichnet wurde. Es zeigt keine Verwandtschaft zu anderen bekannten Proteinen, enthält keine typischen Strukturmotive, wird auf nur einem Exon kodiert und ist in allen bisher untersuchten Vertebraten, einschließlich des Menschen, evolutionär hoch konserviert. Ziel der vorliegenden Arbeit war die nähere funktionelle und proteinbiochemische Charakterisierung Fibins. In vielen Geweben adulter Mäuse (Mus musculus), v. a. in zerebralen und muskulären Proben, konnte Fibin mRNA nachgewiesen werden. Im Vergleich zum Adultus war die Expression in Geweben pränataler Mäuse bedeutend höher und unterschied sich in der Region der Vordergliedmaßen kaum von der im Torso. In L929 (Fibroblasten) und HEK Zellen (embryonale Nierenzellen) wurde eine hohe Expression von Fibin nachgewiesen, die in L929 Zellen durch Glukokortikoide und Aktivatoren des Proteinkinase C / MAP-Kinase , Proteinkinase A sowie des NF-κB / AP-1 bzw. Nrf2 / ARE Signalwegs erhöht werden konnte. Die nicht-proteinkodierende 5‘ Region des humanen Fibin Gens zeigte im Luciferase Reporterassay in L929 und HEK Zellen promotogene Eigenschaften, mit einem Aktivitätsmaximum der Sequenz – 836 Basenpaare bezogen auf den Translationsstartpunkt. In L929 Zellen wurde die promotogene Aktivität durch Glukokortikoide und Aktivatoren des Proteinkinase C / MAP-Kinase- sowie des NF κB / AP 1 bzw. Nrf2 / ARE Signalwegs erhöht. Fibin besitzt eine putative N terminale Signalsequenz und eine N Glykosylierungsstelle, die beide experimentell bestätigt wurden. Rekombinantes Fibin zeigte in der Fluoreszenzmikroskopie in COS-7 Zellen (Fibroblasten) eine hohe Kolokalisation mit dem Endoplasmatischen Retikulum, jedoch nur eine geringe mit dem Golgi Apparat. In COS-7 Zellen wurde es nicht über den sekretorischen Weg freigesetzt und zeigte in proteinbiochemischen Untersuchungen eine hohe Tendenz zur Aggregation und Ausbildung von Disulfidbrücken. Es ist anzunehmen, dass Fibin möglicherweise ein bisher unbekanntes Protein für die Ausbildung von Heteromeren benötigt, um erfolgreich sezerniert zu werden.

Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

The free energy landscape of a protein-like chain is constructed from exhaustive simulation studies using a combination of discontinuous molecular dynamics and parallel tempering methods. The protein model is a repeating sequence of four kinds of monomers, in which hydrogen bond attraction, electrostatic repulsion, and covalent bond vibrations are modeled by step, shoulder and square-well potentials, respectively. These protein-like chains exhibit a helical structure in their folded states. The model allows a natural definition of a configuration by considering which beads are bonded. In the absence of a solvent, the relative free energy of dominant structures is determined from the relative populations, and the probabilities predicted from the calculated free energies are found to be in excellent agreement with the observed probabilities at different temperatures. The free energy landscape of the protein-like chain is analyzed and confirmed to have funnel-like characteristics, confirmed by the fact that the probability of observing the most common configuration approaches unity at low enough temperatures for chains with fewer than 30 beads. The effect on the free energy landscape of an explicit square-well solvent, where the beads that can form intra-chain bonds can also form (weaker) bonds with solvent molecules while other beads are insoluble, is also examined. Simulations for chains of 15, 20 and 25 beads show that at low temperatures, the most likely structures are collapsed helical structures. The temperature at which collapsed helical structures become dominant is higher than in the absence of a solvent. Finally, the dynamics of the protein-like chain immersed in an implicit hard sphere solvent is studied using a simple model in which the implicit solvent interacts on a fast time scale with the chain beads and provides sufficient friction so that the motion of monomers is governed by the Smoluchowski equation. Using a Markovian model of the kinetics of transitions between conformations, the equilibration process from an ensemble of initially extended configurations to mainly folded configurations is investigated at low effective temperatures for a number of different chain lengths. It was observed that folding profiles appear to be single exponentials and independent of temperature at low temperatures.

Protein disulfide isomerases (PDIs) catalyze the oxidation and isomerization of disulfide bonds in proteins passing through the endoplasmic reticulum (ER). Although as many as 20 enzymes are classified as PDI family members, their relative contributions to protein folding have remained an open question. Additionally, Ero1 has been characterized as the ER oxidase that transfers oxidizing equivalents from oxygen to PDI enzymes. However, knockout mice lacking the mammalian Ero1 isoforms, Ero1Lα and Ero1Lβ, are viable, and the role of other potential ER oxidases in maintaining an oxidative ER environment is now an important issue. By systematic depletion of ER PDI family members and potential ER oxidases and assessment of disulfide bond formation of secreted endogenous substrates, I have outlined the functional relationships among some of these enzymes. PDI family member depletion revealed that PDI, although not essential for complete disulfide bond formation in client proteins, is the most significant catalyst of oxidative folding. In comparison, ERp57 acts preferentially on glycosylated substrates, ERp72 functions in a more supplementary capacity, and P5 has no detectable role in formation of disulfide bonds for the substrates assayed. Initially, no impact of depletion of Ero1 was observed under steady state conditions, suggesting that other oxidase systems are working in parallel to support normal disulfide bond formation. Subsequent experiments incorporating a reductive challenge revealed that Ero1 depletion produces the strongest delay in re-oxidation of the ER and oxidation of substrate. Depletion of two other potential ER oxidases, peroxiredoxin 4 (PRDX4) and Vitamin K epoxide reductase (VKOR), showed more modest effects. Upon co-depletion of Ero1 and other oxidases, additive effects were observed, culminating in cell death following combined removal of Ero1, PRDX4, and VKOR activities. These studies affirm the predominant roles of Ero1 in ER oxidation processes and, for the first time, establish VKOR as a significant contributor to disulfide bond formation.