scholarly journals Enhancement of protein stability by an additional disulfide bond designed in human neuroglobin

RSC Advances ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 4172-4179 ◽  
Author(s):  
Hai-Xiao Liu ◽  
Lianzhi Li ◽  
Xin-Zhi Yang ◽  
Chuan-Wan Wei ◽  
Hui-Min Cheng ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Ph Stability ◽  
Structural Alterations

A disulfide bond of Cys120 and Cys15 was rationally designed in human neuroglobin (Ngb) by A15C mutation, which caused minimal structural alterations, whereas enhanced both chemical and pH stability, with a thermal stability higher than 100 °C.

scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

scholarly journals 2P-079 Influences of the disulfide bond of the starch-binding domain of glucoamylase on the protein stability and refolding(The 46th Annual Meeting of the Biophysical Society of Japan)

2008 ◽  
Vol 48 (supplement) ◽  
pp. S87
Author(s):  
Hayuki Sugimoto ◽  
Miho Nakaura ◽  
Shigenori Nishimura ◽  
Shuichi Karita ◽  
Hideo Miyake ◽  
...  
Keyword(s):  
Protein Stability ◽  
Annual Meeting ◽  
Disulfide Bond ◽  
Binding Domain ◽  
Biophysical Society

scholarly journals Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

PLoS ONE ◽  
2014 ◽  
Vol 9 (12) ◽  
pp. e115405 ◽  
Author(s):  
Dan Zabetakis ◽  
Mark A. Olson ◽  
George P. Anderson ◽  
Patricia M. Legler ◽  
Ellen R. Goldman
Keyword(s):  
Thermal Stability ◽  
Disulfide Bond ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Thermal Stability Of

scholarly journals Effect of allotypic variation of human IgG1 on the thermal stability of disulfide-linked knobs-into-holes mutants of the Fc for stable bispecific antibody design

2019 ◽  
Vol 2 (3) ◽  
pp. 65-69
Author(s):  
Hiroki Akiba ◽  
Reiko Satoh ◽  
Satoshi Nagata ◽  
Kouhei Tsumoto
Keyword(s):  
Thermal Stability ◽  
Disulfide Bond ◽  
Antibody Engineering ◽  
Efficient Production ◽  
Affinity Tag ◽  
Scanning Calorimetry ◽  
Disulfide Linkage ◽  
Biophysical Property ◽  
Human Igg1 ◽  
Thermal Stability Of

Abstract Background Disulfide-linked knobs-into-holes (dKiH) mutation is a well-validated antibody engineering technique to force heterodimer formation of different Fcs for efficient production of bispecific antibodies. An artificial disulfide bond is created between mutated cysteine residues in CH3 domain of human IgG1 Fc whose positions are 354 of the “knob” and 349 of the “hole” heavy chains. The disulfide bond is located adjacent to the exposed loop with allotypic variations at positions 356 and 358. Effects of the variation on the biophysical property of the Fc protein with dKiH mutations have not been reported. Methods We produced dKiH Fc proteins of high purity by affinity-tag fusion to the hole chain and IdeS treatment, which enabled removal of mispaired side products. Thermal stability was analyzed in a differential scanning calorimetry instrument. Results We firstly analyzed the effect of the difference in allotypes of the Fcs on the thermal stability of the heterodimeric Fc. We observed different melting profiles of the two allotypes (G1m1 and nG1m1) showing slightly higher melting temperature of G1m1 than nG1m1. Additionally, we showed different characteristics among heterodimers with different combinations of the allotypes in knob and hole chains. Conclusion Allotypic variations affected melting profiles of dKiH Fc proteins possibly with larger contribution of variations adjacent to the disulfide linkage.

scholarly journals In Silico Analysis of Glucose Oxidase from Aspergillus niger: Potential Cysteine Mutation Sites for Enhancing Protein Stability

2021 ◽  
Vol 8 (11) ◽  
pp. 188
Author(s):  
Sirawit Ittisoponpisan ◽  
Itthipon Jeerapan
Keyword(s):  
Thermal Stability ◽  
Aspergillus Niger ◽  
Glucose Oxidase ◽  
Disulfide Bond ◽  
In Silico ◽  
Disulfide Bonds ◽  
Grand Challenge ◽  
Analysis Tool ◽  
Bond Quality ◽  
Protein Database

Glucose oxidase (GOx) holds considerable advantages for various applications. Nevertheless, the thermal instability of the enzyme remains a grand challenge, impeding the success in applications outside the well-controlled laboratories, particularly in practical bioelectronics. Many strategies to modify GOx to achieve better thermal stability have been proposed. However, modification of this enzyme by adding extra disulfide bonds is yet to be explored. This work describes the in silico bioengineering of GOx from Aspergillus niger by judiciously analyzing characteristics of disulfide bonds found in the Top8000 protein database, then scanning for amino acid residue pairs that are suitable to be replaced with cysteines in order to establish disulfide bonds. Next, we predicted and assessed the mutant GOx models in terms of disulfide bond quality (bond length and α angles), functional impact by means of residue conservation, and structural impact as indicated by Gibbs free energy. We found eight putative residue pairs that can be engineered to form disulfide bonds. Five of these are located in less conserved regions and, therefore, are unlikely to have a deleterious impact on functionality. Finally, two mutations, Pro149Cys and His158Cys, showed potential for stabilizing the protein structure as confirmed by a structure-based stability analysis tool. The findings in this study highlight the opportunity of using disulfide bond modification as a new alternative technique to enhance the thermal stability of GOx.

scholarly journals Improved Insights into Protein Thermal Stability: From the Molecular to the Structurome Scale

2016 ◽  
Author(s):  
Fabrizio Pucci ◽  
Marianne Rooman
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Protein Stability ◽  
Large Scale ◽  
Molecular Level ◽  
Temperature Dependent ◽  
Natural Evolution ◽  
Multi Scale ◽  
Large Scale Analysis ◽  
Thermal Stability Of

AbstractDespite the intense efforts of the last decades to understand the thermal stability of proteins, the mechanisms responsible for its modulation still remain debated. In this investigation, we tackle this issue by showing how a multi-scale perspective can yield new insights. With the help of temperature-dependent statistical potentials, we analyzed some amino acid interactions at the molecular level, which are suggested to be relevant for the enhancement of thermal resistance. We then investigated the thermal stability at the protein level by quantifying its modification upon amino acid substitutions. Finally, a large scale analysis of protein stability - at the structurome level - contributed to the clarification of the relation between stability and natural evolution, thereby showing that the mutational profile of thermostable and mesostable proteins differ. Some final considerations on how the multi-scale approach could help unraveling the protein stability mechanisms are briefly discussed.

scholarly journals BPTI folding revisited: switching a disulfide into methylene thioacetal reveals a previously hidden path

2018 ◽  
Vol 9 (21) ◽  
pp. 4814-4820 ◽  
Author(s):  
Reem Mousa ◽  
Shifra Lansky ◽  
Gil Shoham ◽  
Norman Metanis
Keyword(s):  
Protein Structure ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Trypsin Inhibitor ◽  
Biological Function ◽  
Bovine Pancreatic Trypsin Inhibitor ◽  
Folding Mechanism ◽  
Pancreatic Trypsin Inhibitor ◽  
Model Protein

The folding mechanism of the model protein bovine pancreatic trypsin inhibitor was revisited. By switching the solvent exposed disulfide bond with methylene thioacetal we uncovered a hidden pathway in its folding mechanism. In addition, this moiety enhanced protein stability while fully maintaining the protein structure and biological function.

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