Characterization of intra-molecular distances and site-specific dynamics in chemically unfolded barstar: evidence for denaturant-dependent non-random structure

Abstract

The structure and dynamics of the unfolded form of a protein are expected to play critical roles in determining folding pathways. In this study, the urea and guanidine hydrochloride (GdnHCl)-unfolded forms of the small protein barstar were explored by time-resolved fluorescence techniques. Barstar was labeled specifically with thionitrobenzoate (TNB), by coupling it to the thiol side-chain of a cysteine residue at one of the following positions on the sequence: 14, 25, 40, 42, 62, 82 and 89, in single cysteine-containing mutant proteins. Seven intra-molecular distances (R_DA) under unfolding conditions were estimated from measurements of time-resolved fluorescence resonance energy transfer between the donor Trp53 and the non-fluorescent acceptor TNB coupled to one of the seven cysteine side-chains. The unfolded protein chain expands with an increase in the concentration of the denaturants. The extent of expansion was found to be non-uniform, with different intra-molecular distances expanding to different extents. In general, shorter distances were found to expand less when compared to longer spans. The extent of expansion was higher in the case of GdnHCl when compared to urea. A comparison of the measured values of R_DA with those derived from a model based on excluded volume, revealed that while shorter spans showed good agreement, the experimental values of R_DA of longer spans were smaller when compared to the theoretical values. Sequence-specific flexibility of the polypeptide was determined by time-resolved fluorescence anisotropy decay measurements on acrylodan or 1,5-IAEDANS labeled single cysteine-containing proteins under unfolding conditions. Rotational dynamics derived from these measurements indicated that the level of flexibility increased with increase in the concentration of denaturants and showed a graded increase towards the C-terminal end. Taken together, these results appear to indicate the presence of specific non-random coil structures and show that the deviation from random coil structure is different for the two denaturants.