Molecular crowding in living cells is believed to influence diffusion processes, intermolecular interactions, protein folding, and intracellular transport.  Here, we investigate crowding effects on the rotational and translation diffusion of Rhodamine green (RhG) and enhance green fluorescent protein (EGFP) as compared with homogeneous solvents (buffer and glycerol).  Synthetic macromolecules (Ficoll-70 and Ficoll-400) and proteins (bovine serum albumin, BSA, ovalbumin) were used as crowding agents.  Time-resolved fluorescence anisotropy (picoseconds to nanoseconds) and fluorescence correlation spectroscopy (microseconds - seconds) were used to quantify rotational and translational diffusion coefficients of RhG and EGFP in biomimetic crowded environments.  Using Stock-Einstein model, the rotational-to-translational diffusion coefficient ratio of RhG and EGFP indicate that the non-specific binding and deviation from Brownian diffusion depend on the type of crowding agents (protein versus Ficoll).  These results provide new insights into crowding effects on diffusion and nonspecific binding of fluorophores on multiple scales of both time and concentrations.

Multiscale diffusion of single molecules in biomimetic crowding using multimodal fluorescence spectroscopy

245th National Meeting (2013)

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