scholarly journals Revisiting the Formation of a Native Disulfide Bond: Consequences for Protein Regeneration and Beyond

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5337
Author(s):  
Mahesh Narayan
Keyword(s):  
Protein Folding ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Active Form ◽  
Rnase A ◽  
Oxidative Folding ◽  
Regeneration Rate ◽  
Oxidative Protein Folding ◽  
Pancreatic Ribonuclease A ◽  
The Impact

Oxidative protein folding involves the formation of disulfide bonds and the regeneration of native structure (N) from the fully reduced and unfolded protein (R). Oxidative protein folding studies have provided a wealth of information on underlying physico-chemical reactions by which disulfide-bond-containing proteins acquire their catalytically active form. Initially, we review key events underlying oxidative protein folding using bovine pancreatic ribonuclease A (RNase A), bovine pancreatic trypsin inhibitor (BPTI) and hen-egg white lysozyme (HEWL) as model disulfide bond-containing folders and discuss consequential outcomes with regard to their folding trajectories. We re-examine the findings from the same studies to underscore the importance of forming native disulfide bonds and generating a “native-like” structure early on in the oxidative folding pathway. The impact of both these features on the regeneration landscape are highlighted by comparing ideal, albeit hypothetical, regeneration scenarios with those wherein a native-like structure is formed relatively “late” in the R→N trajectory. A special case where the desired characteristics of oxidative folding trajectories can, nevertheless, stall folding is also discussed. The importance of these data from oxidative protein folding studies is projected onto outcomes, including their impact on the regeneration rate, yield, misfolding, misfolded-flux trafficking from the endoplasmic reticulum (ER) to the cytoplasm, and the onset of neurodegenerative disorders.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

2020 ◽  
Author(s):  
Reem Mousa ◽  
Taghreed Hidmi ◽  
Sergei Pomyalov ◽  
Shifra Lansky ◽  
Lareen Khouri ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Crystal Structure Analysis ◽  
Folding Rate ◽  
Oxidative Folding ◽  
Functional Studies ◽  
Oxidative Protein Folding ◽  
And Function

<p>The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of <i>in vitro</i> oxidative folding of many proteins.</p><p>In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.</p><p>A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. </p>Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

2020 ◽  
Author(s):  
Reem Mousa ◽  
Taghreed Hidmi ◽  
Sergei Pomyalov ◽  
Shifra Lansky ◽  
Lareen Khouri ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Crystal Structure Analysis ◽  
Folding Rate ◽  
Oxidative Folding ◽  
Functional Studies ◽  
Oxidative Protein Folding ◽  
And Function

<p>The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of <i>in vitro</i> oxidative folding of many proteins.</p><p>In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.</p><p>A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. </p>Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

scholarly journals Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins

2015 ◽  
Vol 90 (6) ◽  
pp. 2884-2894 ◽  
Author(s):  
Eden P. Go ◽  
Albert Cupo ◽  
Rajesh Ringe ◽  
Pavel Pugach ◽  
John P. Moore ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Protein A ◽  
Native Conformation ◽  
Purification Method ◽  
Immunodeficiency Virus ◽  
Monomeric Gp120 ◽  
Env Protein ◽  
The Impact ◽  
Hiv 1

ABSTRACTWe investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505envgene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds.IMPORTANCEIt is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

scholarly journals Balanced Ero1 activation and inactivation establishes ER redox homeostasis

2012 ◽  
Vol 196 (6) ◽  
pp. 713-725 ◽  
Author(s):  
Sunghwan Kim ◽  
Dionisia P. Sideris ◽  
Carolyn S. Sevier ◽  
Chris A. Kaiser
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Direct Reduction ◽  
Redox Homeostasis ◽  
Feedback Regulation ◽  
Redox Balance ◽  
Oxidative Protein Folding ◽  
Reduced And Oxidized Glutathione ◽  
Minimum Number ◽  
Substrate Proteins

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1p-mediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding.

Small molecule diselenide additives for in vitro oxidative protein folding

2016 ◽  
Vol 52 (16) ◽  
pp. 3336-3339 ◽  
Author(s):  
Post Sai Reddy ◽  
Norman Metanis
Keyword(s):  
Protein Folding ◽  
Small Molecule ◽  
Oxidative Folding ◽  
Oxidative Protein Folding

Small molecule diselenides were prepared and found to enhance thein vitrooxidative folding of disulfide-rich protein.

scholarly journals Structure of Ero1p, Source of Disulfide Bonds for Oxidative Protein Folding in the Cell

Cell ◽  
2004 ◽  
Vol 117 (5) ◽  
pp. 601-610 ◽  
Author(s):  
Einav Gross ◽  
David B Kastner ◽  
Chris A Kaiser ◽  
Deborah Fass
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Oxidative Protein Folding

scholarly journals Oxidative Folding of Proteins: The “Smoking Gun” of Glutathione

2021 ◽  
Vol 22 (18) ◽  
pp. 10148
Author(s):  
Alessio Bocedi ◽  
Giada Cattani ◽  
Giorgia Gambardella ◽  
Linda Schulte ◽  
Harald Schwalbe ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Protein Folding ◽  
Criminal Trial ◽  
Low Molecular Weight ◽  
Oxidative Folding ◽  
Oxidative Protein Folding

Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found “not guilty”. Now, new surprising evidence repeats its request to be the “smoking gun” which reopens the criminal trial revealing the crucial involvement of this tripeptide.

scholarly journals Oxidative protein folding in eukaryotes

2004 ◽  
Vol 164 (3) ◽  
pp. 341-346 ◽  
Author(s):  
Benjamin P. Tu ◽  
Jonathan S. Weissman
Keyword(s):  
Protein Folding ◽  
Molecular Oxygen ◽  
Disulfide Bonds ◽  
Terminal Electron Acceptor ◽  
Oxidative Protein Folding ◽  
Disulfide Formation ◽  
Open Questions ◽  
The Many ◽  
Related Proteins ◽  
Protein Disulfide

The endoplasmic reticulum (ER) provides an environment that is highly optimized for oxidative protein folding. Rather than relying on small molecule oxidants like glutathione, it is now clear that disulfide formation is driven by a protein relay involving Ero1, a novel conserved FAD-dependent enzyme, and protein disulfide isomerase (PDI); Ero1 is oxidized by molecular oxygen and in turn acts as a specific oxidant of PDI, which then directly oxidizes disulfide bonds in folding proteins. While providing a robust driving force for disulfide formation, the use of molecular oxygen as the terminal electron acceptor can lead to oxidative stress through the production of reactive oxygen species and oxidized glutathione. How Ero1p distinguishes between the many different PDI-related proteins and how the cell minimizes the effects of oxidative damage from Ero1 remain important open questions.

scholarly journals Oxidative Protein-Folding Systems in Plant Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Yayoi Onda
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
De Novo ◽  
Protein Body ◽  
Structural Diversity ◽  
Carrier Proteins ◽  
Exchange Reactions ◽  
Disulfide Exchange ◽  
Protein Storage ◽  
Oxidative Protein Folding

Plants are unique among eukaryotes in having evolved organelles: the protein storage vacuole, protein body, and chloroplast. Disulfide transfer pathways that function in the endoplasmic reticulum (ER) and chloroplasts of plants play critical roles in the development of protein storage organelles and the biogenesis of chloroplasts, respectively. Disulfide bond formation requires the cooperative function of disulfide-generating enzymes (e.g., ER oxidoreductase 1), which generate disulfide bonds de novo, and disulfide carrier proteins (e.g., protein disulfide isomerase), which transfer disulfides to substrates by means of thiol-disulfide exchange reactions. Selective molecular communication between disulfide-generating enzymes and disulfide carrier proteins, which reflects the molecular and structural diversity of disulfide carrier proteins, is key to the efficient transfer of disulfides to specific sets of substrates. This review focuses on recent advances in our understanding of the mechanisms and functions of the various disulfide transfer pathways involved in oxidative protein folding in the ER, chloroplasts, and mitochondria of plants.

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