NMR-Detected Brownian Dynamics of α B-Crystallin over a Wide Range of Concentrations

Abstract

Knowledge about the global translational and rotational motion of proteins under crowded conditions is highly relevant for understanding the function of proteins in vivo. This holds in particular for human αB-crystallin, which is strongly crowded in vivo and inter alia responsible for preventing cataracts. Quantitative information on translational and rotational diffusion is not readily available, and we here demonstrate an approach that combines pulsed-field-gradient NMR for translational diffusion and proton T1ρ/T2 relaxation-time measurements for rotational diffusion, thus overcoming obstacles encountered in previous studies. The relaxation times measured at variable temperature provide a quantitative measure of the correlation function of protein tumbling, which cannot be approximated by a single exponential, because two components are needed for a minimal and adequate description of the data. We find that at high protein concentrations, rotational diffusion is decoupled from translational diffusion, the latter following the macroscopic viscosity change almost quantitatively, resembling the behavior of spherical colloids. Analysis of data reported in the literature shows that well-packed globular proteins follow a scaling relation between the hydrodynamic radius and the molar mass, Rh ∼ M(1/d), with a fractal dimension of d ∼ 2.5 rather than 3. Despite its oligomeric nature, Rh of αB-crystallin as derived from both NMR methods is found to be fully consistent with this relation.

NMR of sedimented, fibrillized, silica-entrapped and microcrystalline (metallo)proteins

“…Interparticle interactions can affect to different extents the translational and the rotational diffusion. On this topic, the reader is referred to the discussion given by Krushelnitsky in [76] and by Roos et al in [136].…”

Immobilization of soluble protein complexes in MAS solid-state NMR: Sedimentation versus viscosity

“…At very high concentrations globular proteins typically retain a high degree of rotational mobility as they can rotate rather freely within a cage. It is found that the correlation time of rotational diffusion τ R is proportional to the molecular weight M W following the relation τ R (µs) ~ M W 3/d (kDa), with d being approximately d=2.5 [53]. Depending on temperature, a rotational correlation time τ R on the order of 1.2-5.7 µs was found for αB-crystallin [9,53].…”

“…Molecular crowding impacts both translational and rotational diffusion of a protein. Whereas translational diffusion is largely determined by viscosity, intermolecular protein-protein interactions have a much smaller effect on rotational diffusion [53]. At very high concentrations globular proteins typically retain a high degree of rotational mobility as they can rotate rather freely within a cage.…”

Transient binding accounts for apparent violation of the generalized Stokes–Einstein relation in crowded protein solutions

“…Provided that only the viscosity depends on the concentration c of the dispersed particles but not the particle's (apparent) hydrodynamic size, long-time translational diffusion coefficients may thus be expected to follow the same concentration dependence as the inverse viscosity of the same solution. 24,29 For proteins under macromolecular crowding the applicability of the GSE relationship is either confirmed 10,30,31 or shown to be invalid, 31,32 depending on the specific proteins and crowding agents used. 28 However, for charge-stabilized colloids deviations have been noticed.…”