Background: Extensive studies of peptide conformation have provided reasonable knowledge of the rules determining helix stability. This knowledge can be used to stabilize proteins against chemical and thermal denaturation. This has been done in two proteins: the chemotactic protein from Escherichia coli, Che Y (a 129 aa alpha/beta parallel protein with five alpha-helices, which shows an accumulating intermediate during refolding) and the activation domain of human procarboxypeptidase A2, ADA2h (a 81 aa alpha+beta protein domain, with two alpha-helices, which follows a two-state mechanism). As the introduced stabilizing interactions are local in nature, the energy balance between the contribution of local and nonlocal interactions changes considerably. Recent theoretical analyses of protein folding using simplified models have indicated that optimization of folding speed requires this balance to be biased towards nonlocal interactions. To determine whether this is the case, we study here the folding kinetics of two ADA2h mutants in which alpha-helix 1 (mutant M1) or 2 (mutant M2) has been stabilized through local interactions, as well as the equilibrium and kinetic behaviour of a double mutant (DM) in which both helices have been stabilized. Results: The stability of DM is considerably enhanced with respect to wild type (WT) and this mutant can be considered as a thermoresistant protein (T-m > 363 K). The thermodynamic parameters obtained by chemical denaturation (urea and GdnHCl) show that DM is similar to 2.6 kcal mol(-1) more stable than WT. The effects on folding kinetics are different in each of the single mutants. MI shows very little effect in refolding, while its unfolding is greatly decelerated with respect to WT. M2 shows, together with a deceleration in unfolding, a significant acceleration in refolding. As with equilibrium parameters, the kinetics of the double mutant can be explained by the simple addition of the effects found in each single mutant. Interestingly enough, the refolding slope m(kf) in mutants M2 and DM is smaller than in the wild-type and M1 mutant. Conclusions: Thermoresistance can be achieved, in some cases, by increasing favourable native local interactions. The balance between local and nonlocal interactions can be significantly changed in some proteins and still keep a cooperative unfolding transition similar to that of the wild type. The introduction of favourable local interactions by mutational redesign can also be used to increase the folding speed of certain proteins, showing that not all proteins in nature have been optimized for rapid folding, contrary to what has been theoretically indicated. This behaviour is probably also shared by other polypeptides with highly unstructured denatured states. All these phenomena have been shown experimentally in ADA2h by mutations that increase helix stability. However, the effects promoted for such an approach in proteins with residual structure and/or intermediates in the denatured ensemble could be different. This has been shown by experiments performed on CheY in which the cooperativity of the folding process was greatly affected.
