COMPACTNESS OF PROTEIN MOLTEN GLOBULES - TEMPERATURE-INDUCED STRUCTURAL-CHANGES OF THE APOMYOGLOBIN FOLDING INTERMEDIATE

Apomyoglobin undergoes a two-step unfolding transition when the pH is lowered from 6 to 2. The partly folded intermediate (I) state at pH 4 and low ionic strength has properties of a molten globule. We have stu died structural features of this state, its compactness, content of se condary structure, and specific packing of aromatic side chains, using dynamic light scattering, and small-angle X-ray scattering and far- a nd near-ultraviolet circular dichroism spectroscopy. Particular attent ion was paid to temperature-dependent structural changes. The results are discussed with reference to the native-like (N) state and the high ly unfolded (U) state. It turned out that the I-state is most compact near 30 degrees C, having a Stokes radius 20% larger and a radius of g yration 30% larger than those of the N-state. Both cooling and heating relative to 30 degrees C led to an expansion of the molecule, but the structural changes at low and high temperatures were of a different k ind. At temperatures above 40 degrees C non co-operative melting of st ructural elements was observed, while the secondary structure was esse ntially retained on cooling. The results are discussed in context with theoretical predictions of the compactness and the stability of apomy oglobin by Alonso et al. [Alonso, D. O. V., Dill, K. A., and Stigter, D. (1991) Biopolymers 31:1631-1649]. Comparing the I-state of apomyogl obin with the molten globules of alpha-lactalbumin and cytochrome c, w e found that the compactness of the molten globule states of the three proteins decreases in the order alpha-lactalbumin > apocytochrome c > apomyoglobin. While alpha-lactalbumin and cytochrome c are rather hom ogeneously expanded, apomyoglobin exhibits a non uniform expansion, si nce two structural domains could clearly be detected by small-angle X- ray scattering.