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Engineering ClpS for Selective and Enhanced N-terminal Amino Acid Binding



Jennifer A. Tullman, Zvi Kelman, John P. Marino


One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity, sequential recognition and detection of specific amino acids that comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-End Rule Pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we have employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, two variants of the Agrobacterium tumefaciens ClpS protein with native residues 34-36 ProArgGlu mutated to ProMetSer and CysProSer were identified. Using in vitro surface binding assays, the ProMetSer variant was found to have enhanced affinity for Phe at the N-terminus with 7-fold tighter binding relative to wild-type ClpS; while, the CysProSer variant is shown to bind selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe.
Applied Microbiology and Biotechnology


directed evolution, yeast display, amino acid binding, peptide sequencing


Tullman, J. , Kelman, Z. and Marino, J. (2019), Engineering ClpS for Selective and Enhanced N-terminal Amino Acid Binding, Applied Microbiology and Biotechnology, [online], (Accessed June 19, 2024)


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Created March 1, 2019, Updated February 13, 2020