Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii

Yin, Xin; Hu, Die; Li, Jianfang; He, Yao; Zhu, Tian-Di; Wu, Minchen

doi:10.1371/journal.pone.0126864

Cited by 36 publications

(17 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Here we show that this same region can be cross-linked with a long disulfide bond, which results in a 9°C increase in thermal stability. A survey of the literature reveals that most engineered cysteine disulfides give a Tm increase around 5°C, although there are a few cases where a more than 10°C increase in Tm is observed (27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Interestingly, in work related to that described here, Xiang et al (13) demonstrated that halogen-containing NCAAs with relatively long side chains are capable of forming a covalent linkages with a Cys residue and showed that they improved the thermal stability of an affibody by 13°C in vitro.…”

Section: Discussionmentioning

confidence: 73%

Enhancing protein stability with extended disulfide bonds

Liu

Wang

Luo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

158

View full text Add to dashboard Cite

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9°C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.noncanonical amino acids | extended disulfide bonds | β-lactamase | thermostability | evolutionary advantage

show abstract

Section: Discussionmentioning

confidence: 73%

Enhancing protein stability with extended disulfide bonds

Liu

Wang

Luo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

158

View full text Add to dashboard Cite

show abstract

“…The introduction of disulfide bonds in protein structure is considered as a common strategy to improve the thermostability of industrial enzymes [ 36 – 38 ], however, incorrect design of disulfide bonds may also reduce the catalytic activity and thermostability [ 39 – 41 ]. A careful selection of the residue pairs in enzyme structure for disulfide bonds engineering was required.…”

Section: Discussionmentioning

confidence: 99%

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis

Niu

Zhu

et al. 2016

PLoS ONE

View full text Add to dashboard Cite

1,3–1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C) were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm) compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

show abstract

“…Considering also the importance of disulphide bonds for the maintenance of enzyme conformation (considering in this case plasma esterases) and the impact of heat on disulphide bonds, three aliquots of QCs that underwent thermal inactivation were then kept for 24 h on the benchtop at RT and finally compared with freshly extracted QCs. 22,23…”

Section: Recovery (Rec) and Extraction Efficiency (Ee)mentioning

confidence: 99%

Development and validation of a UHPLC-MS/MS method for quantification of the prodrug remdesivir and its metabolite GS-441524: a tool for clinical pharmacokinetics of SARS-CoV-2/COVID-19 and Ebola virus disease

Avataneo

Nicolò

Cusato

et al. 2020

Journal of Antimicrobial Chemotherapy

View full text Add to dashboard Cite

Background: Remdesivir has received significant attention for its potential application in the treatment of COVID-19, caused by SARS-CoV-2. Remdesivir has already been tested for Ebola virus disease treatment and found to have activity against SARS and MERS coronaviruses. The remdesivir core contains GS-441524, which interferes with RNA-dependent RNA polymerases alone. In non-human primates, following IV administration, remdesivir is rapidly distributed into PBMCs and converted within 2 h to the active nucleoside triphosphate form, while GS-441524 is detectable in plasma for up to 24 h. Nevertheless, remdesivir pharmacokinetics and pharmacodynamics in humans are still unexplored, highlighting the need for a precise analytical method for remdesivir and GS-441524 quantification.Objectives: The validation of a reliable UHPLC-MS/MS method for remdesivir and GS-441524 quantification in human plasma.Methods: Remdesivir and GS-441524 standards and quality controls were prepared in plasma from healthy donors. Sample preparation consisted of protein precipitation, followed by dilution and injection into the QSight 220 UHPLC-MS/MS system. Chromatographic separation was obtained through an Acquity HSS T3 1.8 lm, 2.1%50 mm column, with a gradient of water and acetonitrile with 0.05% formic acid. The method was validated using EMA and FDA guidelines.Results: Analyte stability has been evaluated and described in detail. The method successfully fulfilled the validation process and it was demonstrated that, when possible, sample thermal inactivation could be a good choice in order to improve biosafety.Conclusions: This method represents a useful tool for studying remdesivir and GS-441524 clinical pharmacokinetics, particularly during the current COVID-19 outbreak.

show abstract

Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii

Cited by 36 publications

References 45 publications

Enhancing protein stability with extended disulfide bonds

Enhancing protein stability with extended disulfide bonds

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis

Development and validation of a UHPLC-MS/MS method for quantification of the prodrug remdesivir and its metabolite GS-441524: a tool for clinical pharmacokinetics of SARS-CoV-2/COVID-19 and Ebola virus disease

Contact Info

Product

Resources

About