Investigating the Protease Active Site Environment with Vibrational Reporters and X-Ray Crystallography

Abstract

The active sites of proteases were probed utilizing inhibitors modified with vibrational reporters and protein X-ray crystallography. Specifically, the inhibitors 1-cyano-benzene-4-sulfonylfluoride (CBSF) and 1-azido-benzene-4-sulfonylfluoride (ABSF) were synthesized containing the nitrile and azide vibrational reporters, respectively and reacted with the serine proteases subtilisin and trypsin to probe the active sites of the enzymes. Protein crystal structures of a number of these novel protein-inhibitor complexes have been determined using X-ray crystallography to illustrate successful incorporation of these inhibitors exclusively into the active site of these enzymes. This work shows that the tertiary structure of the active site closely matches that of previously published inhibitor-bound protease structures. Details about the X-ray crystal structures of the various protein-inhibitor complexes will be discussed. Complementary infrared spectroscopic results will also be presented to illustrate the ability of this combined approach to effectively probe the active site of proteases with inhibitors containing vibrational reporters.