Comparative methodologies for protein-protein interactions of a novel crowding sensor: ensemble versus single-molecule studies

Abstract

Living cells are composed of complex, crowded, and dynamic microenvironments that regulate biological function and survival, yet these mechanisms are not understood. Cellular macromolecular crowding is known to play a significant role in transport, diffusion, reaction kinetics, equilibria, and protein folding. Therefore, quantitative, site-specific, and non-invasive methods have become a critical target for investigating the correlation between macromolecular crowding and cell pathophysiology. Here we investigate a novel genetically encoded fluorescent protein biosensor for observing macromolecular crowding, GE2.3, composed of mEGFP-linker-mScarlet-I, using integrated laser spectroscopy methods towards site-specific studies of macromolecular crowding in living cells or tissues. The spectroscopic characteristics of GE2.3 are advantageous for minimal interference with intrinsic cellular autofluorescence. The environmental sensitivity of GE2.3 can be quantified using Förster resonance energy transfer (FRET) from the donor molecule (mEGFP) to the acceptor molecule (mScarlet-I), which is sensitive to donor-acceptor distance. Using time-resolved fluorescence and time-resolved fluorescence depolarization, we characterized the photophysical, excited state, and molecular dynamics of GE2.3 for ensemble-based macromolecular crowding studies. Complementary fluorescence correlation spectroscopy studies of GE2.3 also provide a single-molecule assessment of environmental sensitivity. Our results indicate that GE2.3 is sensitive to mimetic crowding as a function of the polymer Ficoll-70 on multiple temporal scales and from single molecule to ensemble level. Additionally, our comparative studies of the translational and rotational dynamics of GE2.3 observed deviations from the Stokes-Einstein model of diffusion. These results in well-defined environments inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of crowded intracellular environment under different physiological conditions.