Verification of sortase for protein conjugation by single-molecule force spectroscopy and molecular dynamics simulations

Sortase is one of the most widely used enzymes for covalent protein conjugation that links protein and protein/small molecules together in a site-specific way. It typically recognizes the "GGG" and "LPXTG" peptide sequences and conjugates them into an "LPXTGGG" linker....

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Bibliographic Details
Published in:Chemical communications (Cambridge, England) England), 2020-04, Vol.56 (28), p.3943-3946
Main Authors: Tian, Fang, Li, Guoqiang, Zheng, Bin, Liu, Yutong, Shi, Shengchao, Deng, Yibing, Zheng, Peng
Format: Article
Language:English
Subjects:
Conjugation
Glycine
Molecular dynamics
Proteins
Simulation
Spectroscopy
Spectrum analysis
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Summary:Sortase is one of the most widely used enzymes for covalent protein conjugation that links protein and protein/small molecules together in a site-specific way. It typically recognizes the "GGG" and "LPXTG" peptide sequences and conjugates them into an "LPXTGGG" linker. As a non-natural linker with several flexible glycine residues, it is unknown whether it affects the properties of the conjugated protein. To verify the use of sortase for protein-protein conjugation, we combined single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to characterize sortase-conjugated polyprotein I27 with three different linkers. We found that the I27 with classic linkers "LPETGGG" and "LPETG" from sortase ligation were of normal stability. However, a protein with a longer artificial linker "LPETGGGG" showed a 15% lower unfolding force. MD simulations revealed that the 4G linker showed a high probability of a closed conformation, in which the adjacent monomer has transient protein-protein interaction. Thus, we verify the use of sortase for protein conjugation, and a longer linker with a higher glycine content should be used with caution. SMFS and MD simulations revealed a closed conformation and a decreased stability of sortase-mediated polyprotein I27 when a linker with a high content of glycine is used.
ISSN:1359-7345
1364-548X
DOI:10.1039/d0cc00714e