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Harnessing the potential of bacterial oxidative folding to aid protein production.
Slater, SL, Mavridou, DAI
Molecular microbiology. 2021;(1):16-28
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Abstract
Protein folding is central to both biological function and recombinant protein production. In bacterial expression systems, which are easy to use and offer high protein yields, production of the protein of interest in its native fold can be hampered by the limitations of endogenous posttranslational modification systems. Disulfide bond formation, entailing the covalent linkage of proximal cysteine amino acids, is a fundamental posttranslational modification reaction that often underpins protein stability, especially in extracytoplasmic environments. When these bonds are not formed correctly, the yield and activity of the resultant protein are dramatically decreased. Although the mechanism of oxidative protein folding is well understood, unwanted or incorrect disulfide bond formation often presents a stumbling block for the expression of cysteine-containing proteins in bacteria. It is therefore important to consider the biochemistry of prokaryotic disulfide bond formation systems in the context of protein production, in order to take advantage of the full potential of such pathways in biotechnology applications. Here, we provide a critical overview of the use of bacterial oxidative folding in protein production so far, and propose a practical decision-making workflow for exploiting disulfide bond formation for the expression of any given protein of interest.
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Thiol-disulfide homeostasis: an integrated approach with biochemical and clinical aspects.
Erel, Ö, Erdoğan, S
Turkish journal of medical sciences. 2020;(SI-2):1728-1738
Abstract
Dynamic thiol-disulfide homeostasis (TDH) is a new area has begun to attract more scrutiny. Dynamic TDH is reversal of thiol oxidation in proteins and represents the status of thiols (-SH) and disulfides (-S-S-). Organic compounds containing the sulfhydryl group is called thiol, composed of sulfur and hydrogen atoms. Disulfides are the most important class of dynamic, redox responsive covalent bonds build in between two thiol groups. For many years, thiol levels were analyzed by several methods. During last years, measurements of disulfide levels have been analyzed by a novel automated method, developed by Erel and Neselioglu. In this method, addition to thiol (termed as native thiol) levels, disulfide levels were also measured and sum of native thiol and disulfide levels were termed as total thiol. Therefore, TDH was begun to be understood in organism. In healthy humans, TDH is maintained within a certain range. Dysregulated dynamic TDH has been implicated several disorders with unknown etiology. A growing body of evidence has demonstrated that the thiol-disulfide homeostasis is involved in variety diseases, such as diabetes mellitus, hypertension, nonsmall cell lung cancer, familial Mediterranean fever (FMF), inflammatory bowel diseases, occupational diseases, gestational diabetes mellitus and preeclampsia. These results may elucidate some pathogenic mechanism or may be a predictor indicating diagnostic clue, prognostic marker or therapeutic sign. In conclusion, protection of the thiol-disulfide homeostasis is of great importance for the human being. Evidence achieved so far has proposed that thiol-disulfide homeostasis is an important issue needs to elucidate wholly.
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Protein Disulfide Exchange by the Intramembrane Enzymes DsbB, DsbD, and CcdA.
Bushweller, JH
Journal of molecular biology. 2020;(18):5091-5103
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Abstract
The formation of disulfide bonds in proteins is an essential process in both prokaryotes and eukaryotes. In gram-negative bacteria including Escherichia coli, the proteins DsbA and DsbB mediate the formation of disulfide bonds in the periplasm. DsbA acts as the periplasmic oxidant of periplasmic substrate proteins. DsbA is reoxidized by transfer of reducing equivalents to the 4 TM helix membrane protein DsbB, which transfers reducing equivalents to ubiquinone or menaquinone. Multiple structural studies of DsbB have provided detailed structural information on intermediates in the process of DsbB catalyzed oxidation of DsbA. These structures and the insights gained are described. In proteins with more than one pair of Cys residues, there is the potential for formation of non-native disulfide bonds, making it necessary for the cell to have a mechanism for the isomerization of such non-native disulfide bonds. In E. coli, this is mediated by the proteins DsbC and DsbD. DsbC reduces mis-formed disulfide bonds. The eight-TM-helix protein DsbD reduces DsbC and is itself reduced by cytoplasmic thioredoxin. DsbD also contributes reducing equivalents for the reduction of cytochrome c to facilitate heme attachment. The DsbD functional homolog CcdA is a six-TM-helix membrane protein that provides reducing equivalents for the reduction of cytochrome c. A recent structure determination of CcdA has provided critical insights into how reducing equivalents are transferred across the membrane that likely also provides understanding how this is achieved by DsbD as well. This structure and the insights gained are described.
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Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway.
Robinson, PJ, Bulleid, NJ
Cells. 2020;(9)
Abstract
Disulphide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulphide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulphide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulphide bond formation is reviewed with emphasis on events that occur during ER entry. Disulphide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulphide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulphide bond formation and identifies key questions that need to be addressed to further understand this fundamental process.
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Structures and disulfide cross-linking of de novo designed therapeutic mini-proteins.
Silva, DA, Stewart, L, Lam, KH, Jin, R, Baker, D
The FEBS journal. 2018;(10):1783-1785
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Abstract
Recent advances in computational protein design now enable the massively parallel de novo design and experimental characterization of small hyperstable binding proteins with potential therapeutic activity. By providing experimental feedback on tens of thousands of designed proteins, the design-build-test-learn pipeline provides a unique opportunity to systematically improve our understanding of protein folding and binding. Here, we review the structures of mini-protein binders in complex with Influenza hemagglutinin and Bot toxin, and illustrate in the case of disulfide bond placement how analysis of the large datasets of computational models and experimental data can be used to identify determinants of folding and binding.
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Chemistry and Enzymology of Disulfide Cross-Linking in Proteins.
Fass, D, Thorpe, C
Chemical reviews. 2018;(3):1169-1198
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Abstract
Cysteine thiols are among the most reactive functional groups in proteins, and their pairing in disulfide linkages is a common post-translational modification in proteins entering the secretory pathway. This modest amino acid alteration, the mere removal of a pair of hydrogen atoms from juxtaposed cysteine residues, contrasts with the substantial changes that characterize most other post-translational reactions. However, the wide variety of proteins that contain disulfides, the profound impact of cross-linking on the behavior of the protein polymer, the numerous and diverse players in intracellular pathways for disulfide formation, and the distinct biological settings in which disulfide bond formation can take place belie the simplicity of the process. Here we lay the groundwork for appreciating the mechanisms and consequences of disulfide bond formation in vivo by reviewing chemical principles underlying cysteine pairing and oxidation. We then show how enzymes tune redox-active cofactors and recruit oxidants to improve the specificity and efficiency of disulfide formation. Finally, we discuss disulfide bond formation in a cellular context and identify important principles that contribute to productive thiol oxidation in complex, crowded, dynamic environments.
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Protein secretion and the endoplasmic reticulum.
Benham, AM
Cold Spring Harbor perspectives in biology. 2012;(8):a012872
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Abstract
In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is controlled in the ER is now quite sophisticated. However, there remain gaps in our knowledge, particularly when applying insight gained from model systems to the more complex situations found in vivo. This article describes recent advances in our understanding of the ER and its role in preparing proteins for secretion, with an emphasis on glycoprotein quality control and pathways of disulfide bond formation.
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Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function.
Liu, H, May, K
mAbs. 2012;(1):17-23
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Abstract
The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. Non-classical disulfide bond structure was first identified in IgG4 and later in IgG2 antibodies. Although, cysteine residues should be in the disulfide bonded states, free sulfhydryls have been detected in all subclasses of IgG antibodies. In addition, disulfide bonds are susceptible to chemical modifications, which can further generate structural variants such as IgG antibodies with trisulfide bond or thioether linkages. Trisulfide bond formation has also been observed for IgG of all subclasses. Degradation of disulfide bond through β-elimination generates free sulfhydryls disulfide and dehydroalanine. Further reaction between free sulfhydryl and dehydroalanine leads to the formation of a non-reducible cross-linked species. Hydrolysis of the dehydroalanine residue contributes substantially to antibody hinge region fragmentation. The effect of these disulfide bond variations on antibody structure, stability and biological function are discussed in this review.
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Thiol/Disulfide redox switches in the regulation of heme binding to proteins.
Ragsdale, SW, Yi, L
Antioxidants & redox signaling. 2011;(6):1039-47
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This review focuses on thiol/disulfide redox switches that regulate heme binding to proteins and modulate their activities. The importance of redox switches in metabolic regulation and the general mechanism by which redox switches modulate activity are discussed. Methods are described to characterize heme-binding sites and to assess their physiological relevance. For thiol/disulfide interconversion to regulate activity of a system, the redox process must be reversible at the ambient redox potentials found within the cell; thus, methods (and their limitations) are discussed that can address the physiological relevance of a redox switch. We review recent results that define a mechanism for how thiol/disulfide redox switches that control heme binding can regulate the activities of an enzyme, heme oxygenase-2, and an ion channel, the BK potassium channel. The redox switches on these proteins are composed of different types of Cys-containing motifs that have opposite effects on heme affinity, yet have complementary effects on hypoxia sensing. Finally, a model is proposed to describe how the redox switches on heme oxygenase-2 and the BK channel form an interconnected system that is poised to sense oxygen levels in the bloodstream and to elicit the hypoxic response when oxygen levels drop below a threshold value.
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Disulfides as redox switches: from molecular mechanisms to functional significance.
Wouters, MA, Fan, SW, Haworth, NL
Antioxidants & redox signaling. 2010;(1):53-91
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Abstract
The molecular mechanisms underlying thiol-based redox control are poorly defined. Disulfide bonds between Cys residues are commonly thought to confer extra rigidity and stability to their resident protein, forming a type of proteinaceous spot weld. Redox biologists have been redefining the role of disulfides over the last 30-40 years. Disulfides are now known to form in the cytosol under conditions of oxidative stress. Isomerization of extracellular disulfides is also emerging as an important regulator of protein function. The current paradigm is that the disulfide proteome consists of two subproteomes: a structural group and a redox-sensitive group. The redox-sensitive group is less stable and often associated with regions of stress in protein structures. Some characterized redox-active disulfides are the helical CXXC motif, often associated with thioredoxin-fold proteins; and forbidden disulfides, a group of metastable disulfides that disobey elucidated rules of protein stereochemistry. Here we discuss the role of redox-active disulfides as switches in proteins.