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Cell-free mutant analysis combined with structure prediction of a lasso peptide biosynthetic protein B2
Open accessPreprint — published as 10.1021/acssynbio.2c00176
Abstract
Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble and no experimentally-solved structures are available. Here, we performed a rabid semi-comprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (TfuB2) using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 TfuB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, TfuB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.
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Highly accurate protein structure prediction with AlphaFold
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Metadata
- Authors
- Alfi, A., Popov, A., Kumar, A., Zhang, K. Y. J., Dubiley, S., Severinov, K., & Tagami, S.
- Affiliations
- RIKEN Center for Biosystems Dynamics Research (JP)
- Skolkovo Institute of Science and Technology (RU)
- Corresponding author
- Tagami, Shunsuke
- ORCID
- Venue
- ACS Synthetic Biology
- Publisher
- openRxiv
- Published
- 2022-04-06
- Volume
- 11
- Issue
- 6
- Pages
- 2022-2028
- ISSN
- 2161-5063 (ISSN-L)
- OpenAlex
- W4282937659
- Cited by
- 16
- References
- 1
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 7 · 2023: 4 · 2022: 1
- FWCI
- 2.10
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- ACS Synthetic Biology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- Funders
- Inamori Foundation
- Russian Science Foundation (22-14-00224)
- Ministry of Science and Higher Education of the Russian Federation (075-10-2021-114)
- RIKEN
- Topics
- Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- lasso peptides, Thermobifida fusca, cysteine protease, cell-free transcription-translation, site-directed mutagenesis, AlphaFold2, protein structure prediction, hydrophobic patch, protein-protein interaction, peptide maturation, structure-function relationship, thermophilic actinomycetes
- MeSH terms
- Peptide Hydrolases, Endopeptidases, Peptides, Proteins, Actinobacteria
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