Fluorescence quenching studies of γ-butyrolactone-binding protein (CprB) from streptomyces coelicolor A3(2)

Abstract

Fluorescence spectroscopy is an important analytical tool which is widely employed to study biological systems. This technique can be applied to qualitatively and quantitatively probe protein-ligand interactions primarily because of its sensitivity, selectivity, nondestructive and rapid form of analysis. In this chapter we describe the utility of this technique to establish a label-free, universal screening protocol for putative γ-butyrolactone (GBL) receptors by exploiting the intrinsic fluorescence of a highly conserved tryptophan residue that constitutes the hydrophobic pocket for GBL binding, a unique feature possessed by this family of receptors. Here we demonstrate this technique using a combination of steady-state fluorescence quenching methods and fluorescence lifetime decay kinetics using CprB protein from Streptomyces coelicolor A3(2) as a model system. Interaction data between CprB and two chemically synthesized GBLs involved in quorum sensing, Cp1 and Cp2, have been used as example.