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ID 65955
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Ohnuma, Kyosuke School of Pharmaceutical Sciences, Okayama University
Yamashita, Atsuko School of Pharmaceutical Sciences, Okayama University ORCID Kaken ID publons researchmap
Yasui, Norihisa School of Pharmaceutical Sciences, Okayama University ORCID Kaken ID publons researchmap
Abstract
Single-chain monellin (SCM) is an engineered protein that links the two chains of monellin, a naturally sweet-tasting protein. This protein is an attractive candidate for use as a sugar replacement in food and beverages and has numerous other applications. Therefore, generating SCM mutants with improved stability is an active area of research to broaden the range of its potential applications. In this study, we focused on the Cys41 residue of SCM, which is a single cysteine residue present at a structurally important position. This residue is often substituted with Ser. However, this substitution may destabilize SCM because Cys41 is buried in the hydrophobic core of the protein. Therefore, we designed mutants that substituted Ala, Val, and Leu for this residue, namely C41A, C41V, and C41L. We characterized these three mutants, SCM C41S, and wild type (WT). Differential scanning fluorimetric analysis revealed that substituting Cys41 with Ala or Val increased the thermal stability of SCM, while substitution with Ser or Leu decreased its stability. Determination of the crystal structures of SCM C41A and C41V mutants revealed that the overall structures and main chain structures around the 41st residue of both mutants were almost identical to the WT. On the other hand, the orientations of the amino acid side chains near the 41st residue differed among the SCM variants. Taken together, our results indicate that substituting Cys41 with Ala or Val increases the stability of SCM and provide insight into the structural basis of this improvement.
Keywords
Crystallography
Monellin
Protein Stability
Recombinant Proteins
Note
The version of record of this article, first published in The Protein Journal, is available online at Publisher’s website: http://dx.doi.org/10.1007/s10930-023-10154-0
Published Date
2023-09-22
Publication Title
The Protein Journal
Volume
volume42
Issue
issue6
Publisher
Springer Science and Business Media LLC
Start Page
698
End Page
708
ISSN
1572-3887
Content Type
Journal Article
language
English
OAI-PMH Set
岡山大学
Copyright Holders
© The Author(s) 2023
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DOI
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Related Url
isVersionOf https://doi.org/10.1007/s10930-023-10154-0
License
http://creativecommons.org/licenses/by/4.0/
Citation
Ohnuma, K., Yamashita, A. & Yasui, N. Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin. Protein J 42, 698–708 (2023). https://doi.org/10.1007/s10930-023-10154-0
Funder Name
Japan Society for the Promotion of Science
助成番号
19H02841
21K19084