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. The N-End Rule Pathway adaptor protein ClpS, natively recognizes N-terminal amino acids (NAAs) on a peptide chain, but lacks the necessary selectivity and affinity for peptide sequencing. We have created three new variants of the Agrobacterium tumefaciens ClpS protein with enhanced affinity and selectivity for Phe, Trp, and Tyr at the N-terminus of a peptide chain that enable use in peptide sequencing applications.
The invention is a protein-based N-terminal amino acid (NAA) detection reagent. There are three variations of the reagent that bind different N-terminal amino acids with varying affinity and selectivity. The full sequences of the proteins and the binding affinity for each amino acid are given in the attached manuscript. The proteins are new proteins that were created by mutating the wild-type Agrobacterium tumefaciens strain C58 ClpS2 protein (NCBI Reference Sequence: WP_010972159.1) and selecting for the mutations that improved the affinity and selectivity for the amino acid of interest. The proteins are useful for peptide sequencing in which each amino acid needs to be detected and identified to a location in that amino acid sequence with high accuracy. The new variant proteins can selectively detect Phe, Trp, and Tyr at the N-terminal position of a peptide with higher selectivity and affinity than the natural occurring protein.
Currently, the proteins' stability at room temperature is not optimal for a biotechnology application such as peptide sequencing that may be use carried out at ambient or higher temperatures. We are therefore in the process of improving the stability of the variant proteins through further mutagenesis. Also, although the affinity is theoretically sufficient for fluorescence based peptide sequencing using the current state of the art in single molecule fluorescence microscopy it may also need to be further improved before putting into practice.
There are currently no commercially available protein-based reagents that can detect Phe, Trp, or Tyr with similar affinity and selectivity to this invention.