How strong are side chain interactions in the folding intermediate?

Influence of 12 nonpolar amino acids residues from the hydrophobic core of apomyoglobin on stability of its native state and folding intermediate was studied. Six of the selected residues are from the A, G and H helices; these are conserved in structure of the globin family, although nonfunctional, that is, not involved in heme binding. The rest are nonconserved hydrophobic residues that belong to the B, C, D, and E helices. Each residue was substituted by alanine, and equilibrium pH-induced transitions in apomyoglobin and its mutants were studied by circular dichroism and fluorescent spectroscopy. The obtained results allowed estimating changes in their free energy during formation of the intermediate state. It was first shown that the strength of side chain interactions in the apomyoglobin intermediate state amounts to 15-50% of that in its native state for conserved residues, and practically to 0% for nonconserved residues. These results allow a better understanding of interactions occurring in the intermediate state and shed light on involvement of certain residues in protein folding at different stages.