Single-Molecule Fluorescence Fluctuation Analysis of Structure-Dependent Fluorescent Probes In Crowded Environment

Abstract

Living cells are crowded with dynamic distributions of macromolecules and organelles, which influence protein assembly and folding, molecular transport, and biochemical reactions. Here, we test the hypothesis that the diffusion of single molecules deviates from Brownian motion as described by the Stokes-Einstein model in a manner that depends on the chemical structure of both the diffusing species and the crowding agents as well as the spatio-temporal resolution of the employed analytical methods. Using fluorescence correlation spectroscopy (FCS), we investigated the translational diffusion of size- and structure-dependent fluorescent probes, at the single-molecule level, in homogeneous (glycerol) and heterogeneous (Ficoll-70) environments. Model fluorescent probes used here are rhodamine green, quantum dots, enhanced green fluorescent proteins, and mCerulean3-linker-mCitrine FRET probes with various linker length and flexibility. Complementary rotational diffusion studies using time-resolved anisotropy enable us to assess weak interactions in crowded environments. Overall, the results show negative deviation from predictions of the Stokes-Einstein model in a fluorophore- and environment-dependent manner. In addition, the estimated hydrodynamic radius of the FRET probe in PBS, using FCS and the Perrin equation, increases as the number of the amino acid residues in the linker increases. These studies are essential for quantitative molecular and cellular biophysics using fluorescence- and diffusion-based studies of protein-protein interactions and biomolecular transport.