Loops linking secondary structure elements affect the stability of molten globule intermediate state of apomyoglobin

Apomyoglobin is a protein widely used as a model for studying globular protein folding. This work aimed to test the hypothesis on influence of rigidity and length of loops linking protein secondary structure elements on the stability of molten globule intermediate state. For this purpose, we studied folding/unfolding of mutant apomyoglobin forms with substitutions of proline residues to glycine and with loops elongated by three and six glycine residues. For all the protein forms, denaturation/renaturation kinetic curves at different urea concentrations were obtained, folding/unfolding constants were calculated and dependencies of rate constant logarithms on urea concentrations were plotted. All the data gave an opportunity to calculate free energies of different apomyoglobin states. As a result, the mutations in apomyoglobin loops were demonstrated to have a real effect on intermediate state stability compared to unfolded state.