Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

AUTOR(ES)

Meldal, M

RESUMO

A solid-phase assay for the complete subsite mapping of the active site of endoproteases has been developed. A library of resin-bound protease substrates was synthesized both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer type of resin that allows proteases to diffuse into the interior and perform their catalytic activity. Anthranilic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet mixing of the beads and parallel washing procedures. After treatment with subtilisin Carlsberg, fluorescing beads were collected and subjected to peptide sequencing, affording the preferred sequences, their cleavage bond, and a semiquantitative estimation of the turnover. A statistical distribution of preferred amino acids was obtained for each subsite. The result was compared with data from kinetic studies in solution.

Documentos Relacionados

• Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries
• Evidence for virus-encoded glycosylation specificity.
• Aurora-A site specificity: a study with synthetic peptide substrates
• Analysis of Airborne Actinomycete Spores with Fluorogenic Substrates
• Fluorogenic substrates for differentiation of gram-negative nonfermentative and oxidase-positive fermentative bacteria.