What Kind of Microviscosity Does a Molecule Experience During its Rotational and Translational Diffusion in Crowded Environments?

Abstract

Living cells are known to be crowded with organelles, biomembranes, and macromolecules such as proteins, DNA, RNA, and actin filaments. In such crowded environments it is reasonable to believe that cellular viscosity is heterogeneous, which is likely to influence biomolecular diffusion, protein-protein interactions, protein-substrate interaction, and protein folding. In this contribution, we investigate the difference between bulk viscosity and microviscosity in crowded environments and their effects on both rotational (ps-ns) and translational (ms-s) diffusion of rhodamine green (as a probe) using time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS), respectively. For biomimetic crowding, Ficoll-70, BSA and ovalbumin were used as crowding agents and compared with glycerol-rich solutions as a homogeneous environment. Assuming a Stockes-Einstein model, the microviscosity was calculated using TRFA and FCS, assuming no binding, and the results are compared with the bulk viscosity, which was measured using a conventional viscometer. Our results indicate that the micro- and bulk viscosities in a homogeneous environment like glycerol-rich solutions are similar over the 1-20 cP range. In Ficoll-70, BSA and ovalbumin-crowded environments, the microviscosity differs from the corresponding bulk viscosity, depending on the nature of crowding agents (i.e., proteins versus polymers) and the concentration of crowding agents. These results are discussed in terms of both non-specific binding and heterogeneous viscosity in crowded solutions, which in return provide an apparent deviation from the Stokes-Einstein model (i.e., Brownian diffusion). Our findings provide a foundation for FCS and TRFA-based studies of diffusion and binding of biomolecules in the crowded milieu of living cells.