Structural basis for the aminoacid composition of proteins from halophilic archea.

Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.

Protein stabilization and the Hofmeister effect: the role of hydrophobic solvation.

Using the IGg binding domain of protein L from Streptoccocal magnus (ProtL) as a case study, we investigated how the anions of the Hofmeister series affect protein stability. To that end, a suite of lysine-to-glutamine modifications were obtained and structurally and thermodynamically characterized. The changes in stability introduced with the mutation are related to the solvent-accessible area of the side chain, specifically to the solvation of the nonpolar moiety of the residue. The thermostability for the set of ProtL mutants was determined in the presence of varying concentrations (0-1 M) of six sodium salts from the Hofmeister series: sulfate, phosphate, fluoride, nitrate, perchlorate, and thiocyanate. For kosmotropic anions (sulfate, phosphate, and fluoride), the stability changes induced by the cosolute (encoded in m(3)=deltaDeltaG(0)/deltaC(3)) are proportional to the surface changes introduced with the mutation. In contrast, the m(3) values measured for chaotropic anions are much more independent of such surface modifications. Our results are consistent with a model in which the increase in the solution surface tension induced by the anion stabilizes the folded conformation of the protein. This contribution complements the nonspecific and weak interactions between the ions and the protein backbone that shift the equilibrium toward the unfolded state.