Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of deltaC(p) of protein unfolding.

Cold and heat denaturation of the double mutant Arg 3-->Glu/Leu 66-->Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D (1)H NMR spectroscopy in the temperature range from -12 degrees C in supercooled water up to +70 degrees C. The fraction of unfolded protein, f (u), was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be -11 degrees C and 39 degrees C, respectively.

Water contributes actively to the rapid crossing of a protein unfolding barrier.

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity.

The effects of ionic strength on protein stability: the cold shock protein family.

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins.

Thermodynamics of a diffusional protein folding reaction.

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters.