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

ABSTRACT A partly folded state of hen egg-white lysozyme has been characterized in 50 % DMSO. Low concentrations of DMSO (F10%) have little effect on the overall folded conformation of lysozyme as seen from 1H NMR chemical shift dispersion. At increasing DMSO concentrations (G10%) a cooperative transi-tion of the structure to a new, partially folded state is observed. This transition is essentially complete by D50 % DMSO. NMR studies show an overall decrease in chemical shift disper-sion with marked broadening of many reso-nances. A substantial number of backbone and side chain–side chain NOEs suggests the pres-ence 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 inter-mediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural tran-sition 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 protec-tion factors are high for the helical domain, whereas NH groups in the triple stranded anti-parallel b-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 non-aqueous structure perturbing solvents like DMSO to stabilize partially folded conforma