Dynamics of tryptophan in the histidine-containing phosphocarrier protein of Streptomyces coelicolor: evidence of multistate equilibrium unfolding

The nanosecond dynamics of the single tryptophan, Trp10, of HPr from Streptomyces coelicolor, HPrsc, has been monitored at different pHs. Time-resolved fluorescence methods and DOSY measurements have been used to map the compactness of the protein. At low pHs, where a molten globule-like species has been described, the correlation times from fluorescence showed an abrupt change as the pH was increased. When the protein was folded (above pH 4), two correlation times were observed, which remained practically constant up to pH 9.5. The long correlation time, around 7.5 ns, corresponds to the global rotational motion of the protein, since this value is in agreement with that determined theoretically from hydrodynamic measurements. The short correlation time, around 1.4 ns, must report on fast movements of the protein segment containing the tryptophan residue. On the other hand, fluorescence lifetimes showed the same abrupt change as the correlation times at low pH, but, in addition, a sigmoidal change with a pKa approximately 4.3 was also observed. On the basis of the modeled structure of HPrsc, this last transition could be due to the proximity of Glu12 to Trp10. The changes monitored by the fluorescence lifetimes agree with those observed previously by steady-state fluorescence, CD, and ANS binding experiments. Taken together, these data suggest a multistate equilibrium during folding of HPrsc starting from low pHs.