Transient binding accounts for apparent violation of the generalized Stokes-Einstein relation in crowded protein solutions.
The effect of high concentration, also referred to as crowding conditions, on Brownian motion is of central relevance for the understanding of the physical, chemical and biological properties of proteins in their native environment. Specifically, the simple inverse relationship between the translational diffusion coefficient and the macroscopic solution viscosity as predicted by the generalized Stokes-Einstein (GSE) relation has been the subject of many studies, yet a consensus on its applicability has not been reached. Here, we use isotope-filtered pulsed-field gradient NMR to separately assess the μm-scale diffusivity of two proteins, BSA and an SH3 domain, in mixtures as well as single-protein solutions, and demonstrate that transient binding can account for an apparent violation of the GSE relation. Whereas GSE behavior applies for the single-protein solutions, it does not hold for the protein mixtures. Transient binding behavior in the concentrated mixtures is evidenced by calorimetric experiments and by a significantly increased apparent activation energy of diffusion. In contrast, the temperature dependence of the viscosity, as well as of the diffusivity in single-component solutions, is always dominated by the flow activation energy of pure water. As a practically relevant second result, we further show that, for high protein concentrations, the diffusion of small molecules such as dioxane or water is not generally a suitable probe for the viscosity experienced by the diffusing proteins.
Martin-Luther-Universität Halle-Wittenberg, Institut für Physik, 06120 Halle (Saale), Germany. matthias.roos@physik.uni-halle.de kay.saalwaechter@physik.uni-halle.de.
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Rothe
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Gruber
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Gröger
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Balbach
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Saalwächter
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Roos
D055592:Biophysical Phenomena; D004058:Diffusion; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012399:Rotation; D012996:Solutions / Q000737:chemistry*; D013696:Temperature; D014783:Viscosity; D014867:Water
D011506:Proteins; D012996:Solutions; D014867:Water