Computer‐aided design of a catalyst for Edman degradation utilizing substrate‐assisted catalysis

Abstract

Molecular biology has been revolutionized by the miniaturization and parallelization of DNA sequencing assays previously performed on bulk samples. Many of these technologies rely on biomolecular reagents to facilitate detection, synthesis, or labeling of samples. To aid in the construction of analogous experimental approaches for proteins and peptides, we have used computer-aided design to engineer an enzyme capable of catalyzing the cleavage step of the Edman degradation. We exploit the similarity between the sulfur nucleophile on the Edman reagent and the catalytic cysteine in a naturally occurring protease to adopt a substrate-assisted mechanism for achieving controlled, step-wise removal of N-terminal amino acids. The ability to expose amino acids iteratively at the N-terminus of peptides is a central requirement for protein sequencing techniques that utilize processive degradation of the peptide chain. While this can be easily accomplished using the chemical Edman degradation, achieving this activity enzymatically in aqueous solution removes the requirement for harsh acid catalysis, improving compatibility with low adsorption detection surfaces, such as those used in single molecule assays.

Paving the way to single-molecule protein sequencing

“…Second, each cycle of Edman degradation can take approximately 45 minutes, making the sequencing process extremely slow. Havranek and colleagues are currently working on an alternative approach to Edman degradation in which an enzyme has been designed that is capable of cleaving off amino acids, one at the time, from the protein N-terminal 40 . The use of this enzyme, called edmanase, may allow Edman degradation to proceed under physiological conditions, and potentially at a faster pace.…”

Computational assessment of the feasibility of protonation-based protein sequencing

“…Fluorescent labels on the NAABs can then be used to determine binding events and their duration. In this method [13,14] several (17) NAABs are used consecutively, with each NAAB having different binding affinities for the different terminal amino acids. In this way, two types of information are obtained: (1) which NAABs bind to the terminal amino acid, and (2) how long they were bound.…”

Single Molecule Protein Sequencing Based on the Superspecificity of tRNA Synthetases

“…In particular see [27] and [28] for information on cleaving at the C-and N-terminal with carboxypeptidases and aminopeptidases respectively. Recently an enzyme designed on a computer and named Edmanase that cleaves residues at the N-end of a protein has been described [29]. Naturally occurring proteins that recognize specific N-terminal amino acids have been studied for possible use in peptide sequencing, leading to variants of the adapter protein ClpS that can recognize Nterminal F (Phenylalanine) and W (Tryptophan) [30].…”