Engineering ClpS for selective and enhanced N-terminal amino acid binding
Jennifer Tullman1, Nicholas Callahan2, Benjamin Ellington2
1Institute for Bioscience and Biotechnology Research (IBBR), National Institute of Standards and Technology (NIST) and the University of Maryland (UMD), 9600 Gudelsky Drive, Rockville, MD, 20850, USA. tullmanj@ibbr.umd.edu.
Applied Microbiology and Biotechnology
|January 25, 2019
Summary
Researchers engineered ClpS proteins to improve single-molecule protein sequencing. Directed evolution created variants with enhanced affinity and selectivity for N-terminal amino acids, advancing peptide sequencing technology.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Single-molecule protein sequencing requires high-fidelity amino acid recognition.
- Naturally occurring proteins like ClpS recognize N-terminal amino acids (NAAs).
- Wild-type ClpS has specificity for N-terminal Phe, Trp, Tyr, and Leu, but modest affinity.
Purpose of the Study:
- To engineer ClpS variants with improved affinity and selectivity for specific NAAs.
- To enhance ClpS for potential applications in peptide sequencing.
Main Methods:
- Directed evolution was used to select for improved ClpS variants.
- Mutations were introduced at residues 34-36 of Agrobacterium tumefaciens ClpS.
- In vitro surface binding assays were performed to evaluate binding affinities and selectivities.
Main Results:
- Two ClpS variants, ProMetSer and CysProSer, were identified.
- The ProMetSer variant showed a sevenfold increase in binding affinity for N-terminal Phe.
- The CysProSer variant exhibited selective binding to N-terminal Trp over Phe with increased affinity for both.
Conclusions:
- Engineered ClpS variants demonstrate enhanced affinity and selectivity for specific NAAs.
- These modified ClpS proteins show potential for improving single-molecule peptide sequencing technologies.
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