Living cells are crowded with a wide range of macromolecules and organelles. Such crowding is believed to affect biological processes such as protein folding, biochemical reactions and diffusion, which are essential for cell survival, function, and ultimately biomedical implications. Yet, the role of crowding on these molecular processes is not fully understood. In this contribution, we used polymers (Ficoll-70) and proteins (BSA, ovalbumin) as biomimetic crowding agents as a means to quantify their effect on the diffusion mechanism of a small molecular probe (namely, rhodamine green, RhG). To distinguish between crowding and viscosity effects on diffusion, control studies were carried out on glycerol-enriched buffer under the same experimental conditions. The viscosity of these crowded environments was quantified independently using viscometer and rheology methods. In contrast with glycerol-rich buffers, the rotational diffusion measurements using time-resolved anisotropy indicate binding reaction (specific or transient due to weak interactions) that is dependent on the concentration and type of the crowding agents. These results on the fast time scale guide our modeling of the translational diffusion of RhG using fluorescence correlation spectroscopy (FCS) as a function of crowding agents. Our results on both translational and rotational diffusion studies of RhG indicate deviation from the Stokes-Einstein model as a function of the concentration and type of the crowding agents. Our studies represent a step forward in the collective effort aimed at elucidating the role of macromolecular crowding towards quantitative cell biology.

Macromolecular crowding effects on both translational and rotational diffusion of molecular probes

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.