Time-resolved fluorescence studies of the molten globule state of apomyoglobin.

We have investigated the structure of the molten globule state of horse heart apomyoglobin by energy transfer experiments. The tyrosine residue at position 146 was converted into 3-nitro-tyrosine and distances between this side-chain and the two tryptophanyl side-chains were obtained from the time-resolved tryptophanyl fluorescence decay curve. Since both Trp residues are located in the N-terminal A-helix and the modified Tyr residue is located in the C-terminal H-helix, these measurements give information about this helix-helix distance. The energy transfer experiments provide direct evidence for a close contact between the A-helix and the H-helix in the molten globule state. This gives a very strong indication of the presence of a single near-native hydrophobic cluster in this state, as previously proposed by other authors. The distance distribution suggests that the fluctuations in the compact states have correlation times shorter than 1 ns. The experiments also show larger fluctuations, both in the native state and in the molten globule state. In addition, the tryptophanyl fluorescence anisotropy decay curves have been measured. The results suggest loose tertiary contacts in the molten globule state, which is in good agreement with earlier studies. For the denatured state of apomyoglobin, both techniques indicate an extended random coil structure.