DEVELOPMENT AND USE OF FLUORESCENCE TECHNIQUES FOR THE STUDY OF PROTEIN CONFORMATIONAL CHANGES

Abstract

The main goal of the present project is to study the hinge movement that the peptidase, timet oligopeptidase (TOP) may undergo upon substrate or inhibitor binding. Fluorescence techniques will be used to study this conformational change in great detail. TOP constructs will be prepared equipped with two donor-donor fluorescent probes (Trp residues) or donor-acceptor probes (5-F Trp - p-nitro-phenylalanine) pairs, one at each side of the central deep channel of the TOP structure.Fluorescence experiments, in steady state and time-resolved mode will inform us about the hinge movement in this enzyme and the impact of different substrates and inhibitors on it. The kinetics of the hinge movement will be investigated using the same construct via stopped-flow fluorescence spectroscopy. A better understanding of the hinge movement will allow us to tune the activity of this enzyme and provides a good basis for designing new efficient inhibitors. We foresee that once we successful demonstrate the potential of this approach especially the 5-FTrp - p-nitro-phenylalanine donor-acceptor pair as outlined in this application, this approach will be adopted by other researchers aiming to study protein conformational changes.