Profiling the SubstrateSpecificity of Viral Protease VP4by a FRET-Based Peptide Library Approach

Knowingthesubstrate specificityofaproteaseisusefulindeterminingitsphysiologicalsubstrates, developing robust assays, and designing specific inhibitors against the enzyme. In this work, we report the development ofacombinatorialpeptide librarymethodfor systematicallyprofiling thesubstrate specificityof endopeptidases. A fluorescent donor (Edans) and quencher (Dabcyl) pair was added to the C- and N-termini of a support-bound peptide. Protease cleavage of the peptide removed the N-terminal quencher, resulting in fluorescent beads, which were isolated and individually sequenced by partial Edman degradation and mass spectrometry (PED-MS) to reveal the peptide sequence, as well as the site of proteolytic cleavage. The method was validated with bovine trypsin and Escherichia coli leader peptidase and subsequently applied to determine the substrate specificity of a viral protease, VP4, derived from the blotched snakehead virus (BSNV).Theresults show thatVP4 cleaves peptides with aconsensussequence of(Abu/Ala/Pro)-X-AlaVX, in agreement with the previously observed cleavage sites in its protein substrates. Resynthesis and a solution- phase assay of several representative sequences against VP4 confirmed the library screening results.