Structural changes in the Ras protein revealed by fluorescence spectroscopy

Abstract

The Y32W mutant of the Ras protein which has a tryptophan residue close to the guanine nucleotide binding site is studied using two fluorescence spectroscopic techniques. Two-dimensional mapping of all emission and all fluorescence spectra using excitation–emission spectroscopy (EES) in conjunction with time-resolved laser-induced fluorescence (LIF) is used to analyze and assign the contribution of the different fluorophores to the total fluorescence. Time-resolved LIF is shown to be a method that allows to follow the slight conformational changes of Ras binding to the nucleotides GDP, GTP, or the non-hydrolyzable analogues GppNHp, GppCH₂p and GTP-γS and allows to distinguish between the active and inactive form. Additionally, a variant of the EES technique is used for the investigation of the intrinsic GTPase function of Ras and the determination of kinetic constants for this reaction.