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Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin
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 impr...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer US
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10590300/ https://www.ncbi.nlm.nih.gov/pubmed/37737932 http://dx.doi.org/10.1007/s10930-023-10154-0 |
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author | Ohnuma, Kyosuke Yamashita, Atsuko Yasui, Norihisa |
author_facet | Ohnuma, Kyosuke Yamashita, Atsuko Yasui, Norihisa |
author_sort | Ohnuma, Kyosuke |
collection | PubMed |
description | 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10930-023-10154-0. |
format | Online Article Text |
id | pubmed-10590300 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Springer US |
record_format | MEDLINE/PubMed |
spelling | pubmed-105903002023-10-23 Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin Ohnuma, Kyosuke Yamashita, Atsuko Yasui, Norihisa Protein J Article 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10930-023-10154-0. Springer US 2023-09-22 2023 /pmc/articles/PMC10590300/ /pubmed/37737932 http://dx.doi.org/10.1007/s10930-023-10154-0 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Ohnuma, Kyosuke Yamashita, Atsuko Yasui, Norihisa Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin |
title | Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin |
title_full | Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin |
title_fullStr | Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin |
title_full_unstemmed | Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin |
title_short | Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin |
title_sort | investigating the effect of substituting a single cysteine residue on the thermal stability of an engineered sweet protein, single-chain monellin |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10590300/ https://www.ncbi.nlm.nih.gov/pubmed/37737932 http://dx.doi.org/10.1007/s10930-023-10154-0 |
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