Applied Microbiology and Biotechnology

, Volume 103, Issue 6, pp 2621–2633 | Cite as

Engineering ClpS for selective and enhanced N-terminal amino acid binding

  • Jennifer TullmanEmail author
  • Nicholas Callahan
  • Benjamin Ellington
  • Zvi Kelman
  • John P. Marino
Biotechnologically relevant enzymes and proteins


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 employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the Agrobacterium tumefaciens ClpS protein with native residues 34–36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.


Peptide sequencing Protein engineering Biosensor N-end rule pathway 



The authors thank Courtnay Brand (IBBR/UMD) for synthesizing all the peptides used in this study; Kenneth Class of the UMD MPRI Flow Cytometry Core Facility; and student interns Mildred Devereux, Zaman Nasser, Eleanor Kelman, Caleb Weddington, Morgan O’Brien, Makenzie Christensen, and Kunal Dharmadhikari.

Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.


Jennifer Tullman was supported by a National Institute of Standards and Technology (NIST) National Research Council (NRC) postdoctoral fellowship. This work was supported by a NIST Innovation in Measurement Science Award.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

A provisional patent application has been submitted based on some of the reagents developed in this work.

Supplementary material

253_2019_9624_MOESM1_ESM.pdf (6.6 mb)
ESM 1 (PDF 6719 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute for Bioscience and Biotechnology Research (IBBR)National Institute of Standards and Technology (NIST) and the University of Maryland (UMD)RockvilleUSA
  2. 2.Biomolecular Labeling LaboratoryIBBRRockvilleUSA

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