Intrinsic Dynamics of Protein-Peptide Unbinding

TL;DR

This study investigates the intrinsic unbinding dynamics of a peptide-protein system using a photoswitchable probe and time-resolved fluorescence, revealing barrier-less unbinding occurring on a microsecond timescale.

Contribution

It introduces a novel experimental setup with a photoswitchable moiety and fluorescence measurement to analyze unbinding dynamics of peptide-protein interactions.

Findings

Unbinding occurs on a few hundred microseconds timescale.

One mutant exhibits on-off binding behavior with no detectable binding in one state.

Unbinding is essentially barrier-less and occurs faster than other variants.

Abstract

The dynamics of peptide-protein binding and unbinding of a variant of the RNase S system has been investigated. To initiate the process, a photoswitchable azobenzene moiety has been covalently linked to the S-peptide, thereby switching its binding affinity to the S-protein. Transient fluorescence quenching was measured with the help of a time-resolved fluorometer, which has been specifically designed for these experiments and is based on inexpensive LED's and laser diodes only. One mutant shows on-off behaviour with no specific binding detectable in one of the states of the photoswitch. Unbinding is faster by at least two orders of magnitudes, as compared to other variants of the RNase S system. It is concluded that unbinding is essentially barrier-less in that case, revealing the intrinsic dynamics of the unbinding event, which occurs on a few 100 microseconds timescale in a strongly stretched-exponential manner.