The role of van der Waals (vdW) packing interactions compared to the hydrophobic effect in stabilizing the functional structure of proteins is poorly understood. Here we show, using fluorescence resonance energy transfer, dynamic fluorescence quenching, red-edge excitation shift, and near- and far-UV circular dichroism, that the pH-induced structural perturbation of a multidomain protein leads to the formation of a state in which two out of the three domains have characteristics of dry molten globules, that is, the domains are expanded compared to the native protein with disrupted packing interactions but have dry cores. We quantitatively estimate the energetic contribution of vdW interactions and show that they play an important role in the stability of the native state and cooperativity of its structural transition, in addition to the hydrophobic effect. Our results also indicate that during the pH-induced unfolding, side-chain unlocking and hydrophobic solvation occur in two distinct steps and not in a concerted manner, as commonly believed.

The nature of the initial structural events during the base-induced unfolding of the native (N) state of proteins is poorly understood. Combining site-specific fluorescence resonance energy transfer, size exclusion chromatography, dynamic fluorescence quenching, red-edge excitation shift and circular dichroism spectroscopy, we show here that an early intermediate during the base-induced unfolding of a multidomain protein, i.e., the B form, has features of a dry molten globule. We show that the N ⇌ B transition involves protein expansion and loosening of packing of inter-domain helices near domains I and II without the disruption of intra-domain packing or any change in hydration of the inter-domain region which resembles a molten hydrocarbon. Surprisingly, the disruption of inter-domain packing accounts for 40-45% of the total change in free energy of complete unfolding. Our results show that the disruption of van der Waals packing can be decoupled in different regions of a protein and could occur prior to hydrophobic solvation during base-induced unfolding, challenging the existing notion.

The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an important field of research for the scientific community for the past five decades. Two important intermediate states, namely, wet molten globule (WMG) and dry molten globule (DMG), have emerged as critical milestones during protein folding−unfolding reactions. While much has been discussed about WMGs as a common unfolding intermediate, the evidence for DMGs has remained elusive owing to their near-native features, which makes them difficult to probe using global structural probes. This Review puts together the available literature and new evidence on DMGs to give a broader perspective on the universality of DMGs and discuss their significance in protein folding, function, and disease.