Changes in the apomyoglobin folding pathway caused by mutation of the distal histidine residue

Factors governing the folding pathways and the stability of apomyoglobin ha ve been examined by replacing the distal histidine at position 64 with phen ylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significa ntly more stable than wild-type apoMb. Kinetic refolding studies of this va riant also show a significant difference from wild-type apoMb. The amplitud e of the burst phase ellipticity in stopped-flow CD measurements is increas ed over that of wild-type, an indication that the secondary structure conte nt of the earliest kinetic intermediate is greater in the mutant than in th e wild-type protein. In addition, the overall rate of folding is markedly i ncreased. Hydrogen exchange pulse labeling was used to establish the struct ure of the initial intermediate formed during the burst phase of the H64F m utant. NMR analysis of the samples obtained at different refolding times in dicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic i ntermediate. Replacement of the polar distal histidine residue with a nonpo lar residue of similar size and shape appears to stabilize the E helix in t he early stages of folding due to improved hydrophobic packing. The presenc e of a hydrophilic histidine at position 64 thus exacts a price in the stab ility and folding efficiency of the apoprotein, but this residue is neverth eless highly conserved among myoglobins due to its importance in function.