Separating the Contribution of Translational and Rotational Diffusion to Protein Association

Abstract

The association of two proteins is preceded by a mutual diffusional search in solution. The role of translational and rotational diffusion in this process has been studied theoretically for many years. However, systematic experimental verification of theoretical results is still lacking. We report here measurements of association rates of the proteins beta-lactamase (TEM) and beta-lactamase inhibitor protein (BLIP) in solutions of glycerol and poly(ethylene glycol) of increasing viscosity. We also measured translational and rotational diffusion in the same solutions, using fluorescence correlation spectroscopy and fluorescence anisotropy, respectively. It is found that in glycerol both translational and rotational diffusion rates are inversely dependent on viscosity, as predicted by the classical Stokes-Einstein relations, while the association rate depends nonlinearly on viscosity. In contrast, the association rate depends only weakly on the viscosity of the polymer solutions, which results in a similar weak dependence of k(on) on viscosity. The data are modeled using the theory of diffusion-limited association. Deviations from the theory are explained by a short-range solute-induced repulsion between the proteins in glycerol solution and an attractive depletion interaction generated by the polymers. These results open the way to the creation of a unified framework for all nonspecific effects involved in the protein association process, as well as to better theoretical understanding of these effects. Further, they reflect on the complex factors controlling protein association within the crowded environment of cells and suggest that a high concentration of macromolecules does not significantly impede protein association.

The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells

“…The rotational viscosity is always smaller than the translational viscosity because in a crowded environment the excluded volume effects and depletion are ubiquitous. The depletion layer i.e., a layer around a particle depleted from other particles (e.g., observed for colloids in polymer solutions) leads to non-uniform viscosity around the particle [139][140][141][142][143][144][145][146]. The viscosity changes from the solvent viscosity close to the surface of an object to the solution viscosity in a bulk solution over a short distance of the depletion layer (usually of the order of 0.1 to few nm).…”

Dynamics of Unfolded Polypeptide Chains in Crowded Environment Studied by Fluorescence Correlation Spectroscopy

“…Such behavior has already been observed for diffusion of proteins in crowded environments probed by FCS and anisotropy measurements. [53][54][55][56] The effect can be explained by differences in macroscopic (measured by a viscosimeter) and microscopic (inferred from molecular diffusional mobilities) viscosities of high molecular mass co-solutes. While for sucrose solutions viscosities are microscopically homogeneous and therefore identical to macroscopic viscosities, a high content of macromolecules induces spatial microscopic heterogeneities in solution by generating a network of pore-like cavities.…”

On the Dynamic Nature of the Transition State for Protein–Protein Association as Determined by Double-mutant Cycle Analysis and Simulation

“…2,3 Non-specific collision events may (in a small percent) develop into an encounter complex, from which, following the transition state, the final complex is formed. The transition state was shown to resemble the final structure, albeit prior to desolvation and structural rearrangements.…”