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.
