Cooperativity of folding of the apomyoglobin pH 4 intermediate studied by glycine and proline mutations.

The apomyoglobin pH 4 folding intermediate contains the A, G, and H helices of myoglobin. Helix destabilizing mutations in the A and G helices are used to test whether the pH 4 folding intermediate of apomyoglobin folds cooperatively. Single glycine or proline mutations destabilize the intermediate substantially, showing that intrinsic helix propensities are important for stability of the intermediate. The A and G helices interact to stabilize each other, as shown by the effect of mutations in the G helix on the unfolding of the A helix, which can be monitored by tryptophan fluorescence. Wild type and the most stable mutant unfold in a two-state reaction, as shown by superposition of the unfolding curves measured by two probes (far-UV circular dichroism and Trp fluorescence), while unfolding of the less stable mutants is more complex. Cooperativity and stability of folding are linked also when stabilizing anions (sulphate, perchlorate) are used to adjust stability.