Equilibrium unfolding of mutant apomyoglobins carrying substitutions of conserved nonfunctional residues with alanine

Protein aggregation or misfolding in the cell is connected with many genetic diseases and can result from substitutions in proteins. Substitutions can influence the protein stability and folding rates in both intermediate and native states. The equilibrium urea-induced unfolding was studied for mutant apomyoglobins carrying substitutions of the conserved nonfunctional residues Val10, Trp14, Ile111, Leu115, Met131, and Leu135 with Ala. Conformational transitions were monitored by intrinsic Trp fluorescence and far-UV circular dichroism. Free energy changes upon transition from the native to the intermediate state and from the intermediate to the unfolded state were determined. All substitutions considerably decreased the stability of native apomyoglobin, whereas the effect on the stability of the intermediate state was essentially smaller.