Population Analysis of Folding Intermediates From Time-Resolved and Spectral Fluorescence of Single-Tryptophan Apoflavodoxin

Abstract

The fluorescence of native apoflavodoxin (i.e. flavodoxin without cofactor FMN) from Azotobacter vinelandii has been extensively used to investigate thermal and denaturant-induced protein (un)folding. The protein populates an off-pathway molten globule-like intermediate during its equilibrium (un)folding. Fluorescence of apoflavodoxin arises mainly from its three tryptophans (Trp74, Trp128 and Trp167). With time-resolved fluorescence anisotropy of wild-type apoflavodoxin, tryptophan-tryptophan energy migration has been quantitatively measured to follow distance variations between two tryptophan pairs during apoflavodoxin (un)folding (N.V. Visser et al. (2008) Biophys. J. 95, 2462-2469). In this study we have followed a more general approach to analyze the time-resolved and steady-state fluorescence results of the single Trp74-mutant apoflavodoxin (Trp128 and Trp167 are replaced by phenylalanine), when it is gradually unfolded by addition of increasing amounts of guanidine hydrochloride. Singular value decomposition (SVD) of data matrices has been used to determine the number of species. In both types of experiments SVD shows the presence of three significant independent components. Therefore we can conclude that the three-state model (native, intermediate and unfolded state of the protein) should be used for further analysis. Each set of experimental data was globally analyzed using the three-state model finally yielding the relative concentrations of all species in the denaturation trajectory. The equilibrium thermodynamic properties were then determined from simultaneous and separate fits of the concentrations obtained from time-resolved and steady-state fluorescence data.