As described in the previous years, our studies have led to the hypothesis that after folding of some unit of the amino acid sequences the interatomic interactions would be globally coupled to generate extra energy, resulting in shift of the equilibrium between folding and unfolding in favor of folding. This hypothesis is not being tested with respect to the mechanism and evoluntionary significance. Thus on completing thermodynamic and kinetic analyses of the effect of substitution of invariant leucine 32 and partially invariant leucine 35 of horse cytochrome c we have proposed that leucine 32, tryptophan 59 and the methionine 80-S-heme iron bond are coupled to generate such extra force. The results with substitution of invariant proline 30 and gylcine 34 have also suggested that these residues would have a role in maintaining such coupling. The NMR studies of hydrogen exchange of RNase (10-118) suggest that a) removal of 6 carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant, b) this would, in turn, alter the enthalphy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold, c) in this activated state the molecule would be highly motile. As reported in the previous years, the two permissible regions for cleavage of horse cytochrome c are consistent with two of the three regions where extra amino acid sequences have been inserted during evolution. Thus, to test where this consistency implies conservation of the global coupling we have been investigating the permissible regions with yeast cytochrome c. The results to date indicate that one of the two permissible regions, namely between residues 39 to 55 (in numbering of horse cytochrome c) may be non-permissible for yeast, possibly because of a change of a single amino acid such as change from leucine to methionine at position 64.