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. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, DeltaC(p) of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.

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. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.

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. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.

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. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.