RESOLVING MULTIPLE PROTEIN CONFORMERS IN EQUILIBRIUM UNFOLDING REACTIONS - A TIME-RESOLVED EMISSION SPECTROSCOPIC (TRES) STUDY OF AZURIN

Unlike steady-state spectrofluorimetry, time-resolved emission spectro scopy (TRES) can resolve emissions from fluorophores with similar quan tum yields and overlapping steady-state emission spectra. Time-resolve d emission studies of the protein-intrinsic fluorophore, tryptophan (T rp), can thus potentially be used to examine protein conformational he terogeneity in solution, as well as to investigate the existence of po pulated intermediate structural states in equilibrium unfolding reacti ons of single-tryptophan proteins. Here, the single-Trp copper protein , azurin, is examined in various concentrations of guanidine hydrochlo ride (GdnCl) with its disulphide bond in an intact state. Interestingl y, multiple envelopes of Trp emission are observed in all TRES spectra acquired, instead of just two emission envelopes (corresponding to th e native and unfolded states) expected from two-state unfolding. These envelopes appear to be centred around the same set of emission wavele ngths in different TRES spectra, and only intensities and decay rates vary with the concentration of denaturant used. This suggests that str uctural states representing different levels of exposure of Trp to the aqueous solvent might in fact, be populated at equilibrium during the unfolding of azurin by GdnCl. (C) 1997 Published by Elsevier Science B.V.