Effects of organic solvents on protein structures: observation of a structured helical core in hen egg-white lysozyme in aqueous dimethylsulfoxide

Abstract

A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO (<10%) have little effect on the overall folded conformation of lysozyme as seen from ¹H NMR chemical shift dispersion. At increasing DMSO concentrations (>10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by ~50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain-side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel β-sheet domain are largely solvent-exposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of nonaqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins.