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

Published

July 27, 2023

Author(s)

Zvi Kelman, John Marino, Christina Bergonzo

Abstract

Numerous technologies are currently in development as a next generation protein sequencing platform. Some published approaches make use of side-chain modification to add fluorescent labels and nucleotide tags. One of the challenges in this approach is differentiating between hydrophobic and aromatic amino acids that lack the necessary chemical moieties for covalent modification, such as leucine and phenylalanine. This problem could be addressed by 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 a NAAB with sufficient affinity and specificity for N-terminal leucine. Enriched variants were analyzed and shown to selectively bind peptides with N-terminal leucine with a substantial decrease in the dissociation rate, a key requirement for a NAAB in next generation protein sequencing.

Citation

Protein Engineering Design & Selection

Pub Type

Journals

Download Paper

https://doi.org/10.1093/protein/gzad007

Local Download

Keywords

next gen protein sequencing, ClpS

Citation

Kelman, Z. , Marino, J. and Bergonzo, C. (2023), Contributions from ClpS surface residues in modulating N-terminal peptide binding and their implications for NAAB development, Protein Engineering Design & Selection, [online], https://doi.org/10.1093/protein/gzad007, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=935636 (Accessed October 20, 2025)