Quantifying oxidative folding

Proteins are involved in almost every known biological process. Their immensely diverse pool of functionalities is largely determined by their amino acid sequence, their three-dimensional structure and by additional modifications known as post translational modifications (PTMs). One of these PTMs is the disulfide bond (DSB) which can be formed between two cysteine amino acids via covalent bonding of two sulfur atoms. DSBs can be found in many important proteins such as antibodies and the processes involved in their formation have been intensely studied for decades. During this time, many - mostly qualitative - aspects of their formation and prevalence have been researched. The research described in this thesis combine parts of this immense existing knowledge and elevate our understanding of the quantitative aspects of DSB formation. The thesis introduction summarises much of this existing knowledge and current research trends are further outlined in a published review. In Chapter 1 the quantitative formation of DSBs in Escherichia coli has been modelled in order to describe and predict both host proteome and recombinant protein DSB formation. Chapter 2 expands our understanding of the DSB forming machinery in the important recombinant protein production host Komagataella phaffii (syn. Pichia pastoris). In the final Chapter, protein structure predictions by AlphaFold are used for predicting both qualitative and quantitative DSB levels in several model organisms

A summary

Coinciding with the Open Access Week 2010 we publish a study on the perceptions and usage trends amongst CSIC scientific community as regards Open Access in general and CSIC institutional repository in particular.Peer reviewe

A quantitative interpretation of oxidative protein folding activity in Escherichia coli

Background: Escherichia coli is of central interest to biotechnological research and a widely used organism for producing proteins at both lab and industrial scales. However, many proteins remain difficult to produce efficiently in E. coli. This is particularly true for proteins that require post translational modifications such as disulfide bonds. Results: In this study we develop a novel approach for quantitatively investigating the ability of E. coli to produce disulfide bonds in its own proteome. We summarise the existing knowledge of the E. coli disulfide proteome and use this information to investigate the demand on this organism’s quantitative oxidative folding apparatus under different growth conditions. Furthermore, we built an ordinary differential equation-based model describing the cells oxidative folding capabilities. We use the model to infer the kinetic parameters required by the cell to achieve the observed oxidative folding requirements. We find that the cellular requirement for disulfide bonded proteins changes significantly between growth conditions. Fast growing cells require most of their oxidative folding capabilities to keep up their proteome while cells growing in chemostats appear limited by their disulfide bond isomerisation capacities. Conclusion: This study establishes a novel approach for investigating the oxidative folding capacities of an organism. We show the capabilities and limitations of E. coli for producing disulfide bonds under different growth conditions and predict under what conditions excess capability is available for recombinant protein production

Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione

In the methylotrophic yeast Komagataella phaffii, we identified an ER-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites and a few non-conventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is non-essential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates

Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast

Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of recombinant secretory proteins. Surprisingly, we found significant differences with both the human and S. cerevisiae systems. Specifically, we report an inactive disulfide linked complex formed by K. phaffii Ero1 and Pdi1, similarly to the human orthologs, but not described in yeast before. Furthermore, we show how the interaction between K. phaffii Pdi1 and Ero1 is unaffected by the introduction of unfolded substrate into the system. This is drastically opposed to the previously observed behavior of the human pathway, suggesting a different regulation of the UPR and/or possibly different interaction mechanics between K. phaffii Pdi1 and Ero1

Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable