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 he lices of myoglobin. Helix destabilizing mutations in the A and G helic es are used to test whether the pH 4 folding intermediate of apomyoglo bin folds cooperatively. Single glycine or proline mutations destabili ze the intermediate substantially, showing that intrinsic helix propen sities are important for stability of the intermediate. The A and G he lices interact to stabilize each other, as shown by the effect of muta tions in the G helix on the unfolding of the A helix, which can be mon itored by tryptophan fluorescence. Wild type and the most stable mutan t unfold in a two-state reaction, as shown by superposition of the unf olding curves measured by two probes (far-UV circular dichroism and Tr p fluorescence), while unfolding of the less stable mutants is more co mplex. Cooperativity and stability of folding are linked also when sta bilizing anions (sulphate, perchlorate) are used to adjust stability.