Engineering ClpS for enhanced N-terminal amino acid binding and use in peptide sequencing

Conference Dates

July 14-18, 2019


As different single-molecule protein sequencing technologies emerge, the need for reagents that can selectively recognized and detect amino acids with high affinity has become apparent. Naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs), such as the N-end rule pathway adaptor protein ClpS can be engineered for enhanced affinity and specificity to meet this requirement. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes other residues at the N-terminal position. We employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for NAAs. In addition, we combined these mutations with rationally designed mutations to improve the thermal stability of the protein. The results and their possible implication to peptide sequencing will be presented.

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