SLOW COOPERATIVE FOLDING OF A SMALL GLOBULAR PROTEIN HPR

The folding of an 85-residue protein, the histidine-containing phospho carrier protein HPr, has been studied using a variety of techniques in cluding DSC, CD, ANS fluorescence, and NMR spectroscopy. In both kinet ic and equilibrium experiments the unfolding of HPr can be adequately described as a two-state process which does not involve the accumulati on of intermediates. Thermodynamic characterization of the native and the transition slates has been achieved from both equilibrium and kine tic experiments. The heat capacity change from the denatured state to the transition state (3.2 kJ mol(-1) K-1) is half of the heat capacity difference between the native and denatured states (6.3 kJ mol(-1) K- 1), while the solvent accessibility of the transition state (0.36) ind icates that its compactness is closer to that of the native than that of the denatured state. The high value for the change in heat capacity upon unfolding results in the observation of cold denaturation at mod erate denaturant concentrations. Refolding from high denaturant concen trations is, however, slow. The rate constant of folding in water, k(f )(H2O) (14.9 s(-1)), is small compared to that reported fur other prot eins of similar size under similar conditions. This indicates that ver y fast refolding is not a universal character of small. globular prote ins which fold in the absence of detectable intermediates.