Structure and dynamics of the alpha-lactalbumin molten globule: Fluorescence studies using proteins containing a single tryptophan residue

The fluorescence properties of three variants of alpha -lactalbumin (alpha -LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of sec ondary structure and stability similar to those of the wild type. The fluor escence signal in the native state is dominated by that of W104, with the s ignal of W60 and W118 significantly quenched by the disulfide bonds in thei r vicinity. In the molten globule state, the magnitude of the fluorescence signal of W60 and W118 increases, due to the loss of rigid, specific side c hain packing. In contrast, the magnitude of the signal of W104 decreases in the molten globule state, perhaps due to the protonation of H107 or quench ing by D102 or K108. The solvent accessibilities of individual tryptophan r esidues were investigated by their fluorescence emission maximum and by acr ylamide quenching studies. In the native state, the order of solvent access ibility is as follows: W118 > W60 > W104. This order changes to W60 > W104 > W118 in the molten globule state. Remarkably, the solvent accessibility o f W118 in the alpha -LA molten globule is lower than that in the native sta te. The dynamic properties of the three tryptophan residues were examined b y time-resolved fluorescence anisotropy decay studies. The overall rotation of the molecule can be observed in both the native and molten globule stat es. In the molten globule state, there is an increase in the extent of loca l backbone fluctuations with respect to the native state. However, the fluc tuation is not sufficient to result in complete motional averaging. The thr ee tryptophan residues in the native and molten globule states have differe nt degrees of motional freedom, reflecting the folding pattern and dynamic heterogeneity of these states. Taken together, these studies provide new in sight into the structure and dynamics of the alpha -LA molten globule, whic h serves as a prototype for partially folded proteins.