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 studied structural features of this state, its compactness, content of secondary structure, and specific packing of aromatic side chains, using dynamic light scattering, and small-angle X-ray scattering and far- and near-ultraviolet circular dichroism spectroscopy. Particular attention was paid to temperature-dependent structural changes. The results are discussed with reference to the native-like (N) state and the highly 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 gyration 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 kind. At temperatures above 40 degrees C non co-operative melting of structural elements was observed, while the secondary structure was essentially retained on cooling. The results are discussed in context with theoretical predictions of the compactness and the stability of apomyoglobin by Alonso et al. [Alonso, D. O. V., Dill, K. A., and Stigter, D. (1991) Biopolymers 31:1631-1649]. Comparing the I-state of apomyoglobin with the molten globules of alpha-lactalbumin and cytochrome c, we 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 homogeneously expanded, apomyoglobin exhibits a non uniform expansion, since two structural domains could clearly be detected by small-angle X-ray scattering.