Profiling the substrate specificity of viral protease VP4 by a FRET-based peptide library approach

Biochemistry. 2009 Jun 23;48(24):5753-9. doi: 10.1021/bi900461e.

Abstract

Knowing the substrate specificity of a protease is useful in determining its physiological substrates, developing robust assays, and designing specific inhibitors against the enzyme. In this work, we report the development of a combinatorial peptide library method for systematically profiling the substrate specificity of 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). The results show that VP4 cleaves peptides with a consensus sequence of (Abu/Ala/Pro)-X-Ala downward arrowX, 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Birnaviridae / enzymology
  • Escherichia coli / metabolism
  • Fluorescence Resonance Energy Transfer
  • Molecular Sequence Data
  • Peptide Library*
  • Peptides / chemistry
  • Serine Endopeptidases / chemistry*
  • Serine Endopeptidases / metabolism
  • Substrate Specificity
  • Viral Proteins / chemistry*
  • Viral Proteins / metabolism

Substances

  • Peptide Library
  • Peptides
  • Viral Proteins
  • Serine Endopeptidases