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A Single Amino Acid Substitution Alters ClpS Binding Specificity



Christina Bergonzo, Jennifer A. Tullman, Kunal Dharmadhikari, Emily Samuels, Makenzie Christensen


ClpS is a small protein currently under development as a probe for detecting specific N-terminal amino acids of peptides. In order for ClpS to be used in this manner, it is important to understand the structural reasons for how and why ClpS recognizes specific residues. To understand the specificity of the recognition mechanism of ClpS, all atom molecular dynamics (MD) simulations were conducted on wild type (WT) ClpS, and ClpS mutants predicted to increase solubility by the Protein Repair One Stop Shop (PROSS) algorithm. Per-residue binding free energies were used to predict that Leu to Asn substitution at position 9 will confer specificity for an N-terminal tyrosine over the preferred phenylalanine. Experimental validation of the L9N mutant using yeast-display assay validated the predicted outcome, showing an increase in tyrosine binding over phenylalanine. Predicting other mutations that can stabilize binding for different N-terminal amino acid combinations will enable engineering artificial protein- interaction networks, and the methods used to conduct this study are transferable and useful in drug delivery problems.
Journal of Molecular Biology


molecular dynamics, binding energies, N-terminal


Bergonzo, C. , Tullman, J. , Dharmadhikari, K. , Samuels, E. and Christensen, M. (2020), A Single Amino Acid Substitution Alters ClpS Binding Specificity, Journal of Molecular Biology, [online], (Accessed June 25, 2024)


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Created March 27, 2020, Updated March 23, 2022