Glycerol-slaved 1H-1H NMR cross-relaxation in quasi-native lysozyme.

1H-1H nuclear cross-relaxation experiments have been carried out with lysozyme in variable glycerol viscosity to study intramolecular motion, self-diffusion, and isotropic rigid-body rotational tumbling at 298 K, pH 3.8. Dynamics of intramolecular 1H-1H cross-relaxation rates, the increase in internuclear spatial distances, and lateral and rotational diffusion coefficients all show fractional viscosity dependence with a power law exponent κ in the 0.17-0.83 range. The diffusion coefficient of glycerol Ds with the bulk viscosity itself is non-Stokesian, having a fractional viscosity dependence on the medium viscosity (Ds âˆ¼ Î·-κ, κ ≈ 0.71). The concurrence and close similarity of the fractional viscosity dependence of glycerol diffusion on the one hand, and diffusion and intramolecular cross-relaxation rates of the protein on the other lead to infer that relaxation of glycerol slaves protein relaxations. Glycerol-transformed native lysozyme to a quasi-native state does not affect the conclusion that both global and internal fluctuations are slaved to glycerol relaxation.

Quasi-native transition and self-diffusion of proteins in water-glycerol mixture.

An orderly investigation of the levels of secondary and tertiary structures, kinetics of tertiary structural changes, and self diffusion coefficient of lysozyme and cytochrome c in the 0-70% (weight/volume) range of glycerol is reported. While secondary structural propensity of both proteins is larger in glycerol, results for tertiary structure and translational diffusion coefficient with increasing glycerol provide two contrasting depictions - lysozyme becomes increasingly compact, plausibly due to disulfide bridge constraints, but cytochrome c expands and loses the tertiary structure. The chain expansion and contraction corresponding to loss and reformation of tertiary structure of cytochrome c are ultrafast that occur in the submillisecond bin. Changes in protein conformation appear in as little as 2% glycerol, and the results suggest that glycerol does not unfold the protein but reversibly destabilizes to quasi-native state(s). These observations make one ponder whether results of studies on protein dynamics, relaxation, and conformational substates reported in literature can be associated with native-state properties.