Contributions from ClpS surface residues in modulating N-terminal peptide binding and their implications for NAAB development.

Abstract

Numerous technologies are currently in development for use in next-generation protein sequencing platforms. A notable published approach employs fluorescently-tagged binding proteins to identity the N-terminus of immobilized peptides, in-between rounds of digestion. This approach makes use of N-terminal amino acid binder (NAAB) proteins, which would identify amino acids by chemical and shape complementarity. One source of NAABs is the ClpS protein family, which serve to recruit proteins to bacterial proteosomes based on the identity of the N-terminal amino acid. In this study, a Thermosynechococcus vestitus (also known as Thermosynechococcus elongatus) ClpS2 protein was used as the starting point for direct evolution of an NAAB with affinity and specificity for N-terminal leucine. Enriched variants were analyzed and shown to improve the interaction between the ClpS surface and the peptide chain, without increasing promiscuity. Interestingly, interactions were found that were unanticipated which favor different charged residues located at position 5 from the N-terminus of a target peptide.