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More than 30 years ago, Nozaki and Tanford reported that the pK values for several amino acids and simple substances in 6 M guanidinium chloride differed little from the corresponding values in low salt (Nozaki, Y., and C. Tanford. 1967. J. Am. Chem. Soc. 89:736-742). This puzzling and counter-intuitive result hinders attempts to understand and predict the proton uptake/release behavior of proteins in guanidinium chloride solutions, behavior which may determine whether the DeltaG(N-D) values obtained from guanidinium chloride-induced denaturation data can actually be interpreted as the Gibbs energy difference between the native and denatured states (Bolen, D. W., and M. Yang. 2000. Biochemistry. 39:15208-15216). We show in this work that the Nozaki-Tanford result can be traced back to the fact that glass-electrode pH meter readings in water/guanidinium chloride do not equal true pH values. We determine the correction factors required to convert pH meter readings in water/guanidinium chloride into true pH values and show that, when these corrections are applied, the effect of guanidinium chloride on the pK values of simple substances is found to be significant and similar to that of NaCl. The results reported here allow us to propose plausible guanidinium chloride concentration dependencies for the pK values of carboxylic acids in proteins and, on their basis, to reproduce qualitatively the proton uptake/release behavior for the native and denatured states of several proteins (ribonuclease A, alpha-chymotrypsin, staphylococcal nuclease) in guanidinium chloride solutions. Finally, the implications of the pH correction for the experimental characterization of protein folding energetics are briefly discussed.

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It is widely recognized that enhancement of protein stability is an important biotechnological goal. However, some applications at least, could actually benefit from stability being strongly dependent on a suitable environment variable, in such a way that enhanced stability or decreased stability could be realized as required. In therapeutic applications, for instance, a long shelf-life under storage conditions may be convenient, but a sufficiently fast degradation of the protein after it has performed the planned molecular task in vivo may avoid side effects and toxicity. Undesirable effects associated to high stability are also likely to occur in food-industry applications. Clearly, one fundamental factor involved here is the kinetic stability of the protein, which relates to the time-scale of the irreversible denaturation processes and which is determined to some significant extent by the free-energy barrier for unfolding (the barrier that "separates" the native state from the highly-susceptible-to-irreversible-alterations nonnative states). With an appropriate experimental model, we show that strong environment-dependencies of the thermodynamic and kinetic stabilities can be achieved using robust protein engineering. We use sequence-alignment analysis and simple computational electrostatics to design stabilizing and destabilizing mutations, the latter introducing interactions between like charges which are screened out at high salt. Our design procedures lead naturally to mutating regions which are mostly unstructured in the transition state for unfolding. As a result, the large salt effect on the thermodynamic stability of our consensus plus charge-reversal variant translates into dramatic changes in the time-scale associated to the unfolding barrier: from the order of years at high salt to the order of days at low salt. Certainly, large changes in salt concentration are not expected to occur in biological systems in vivo. Hence, proteins with strong salt-dependencies of the thermodynamic and kinetic stabilities are more likely to be of use in those cases in which high-stability is required only under storage conditions. A plausible scenario is that inclusion of high salt in liquid formulations will contribute to a long protein shelf-life, while the lower salt concentration under the conditions of the application will help prevent the side effects associated with high-stability which may potentially arise in some therapeutic and food-industry applications. From a more general viewpoint, this work shows that consensus engineering and electrostatic engineering can be readily combined and clarifies relevant aspects of the relation between thermodynamic stability and kinetic stability in proteins.