Rotational dynamics-based assessment of energy transfer efficiency of hetero-FRET probes in crowded environments

Abstract

A living cell is crowded with various organelles, DNA, and proteins. Such macromolecular crowding has a significant impact on cellular processes. Yet, the effects of macromolecular crowding on protein diffusion, reaction rates, and folding are far from understood. As a result, there is a need to quantify crowding in a heterogeneous environment both in vivo and in vitro. Recently, a series of novel genetically encoded FRET probes were developed as sensors to quantitatively measure crowding in vivo and were characterized with steady-state fluorescence (Nat Meth [2015] 12:227). In a crowded environment, these FRET probes are hypothesized to become confined and more compact, thereby leading to enhanced energy transfer. Consequently, the level of crowding can be quantified based on the energy transfer efficiency of the probes. In this Thesis, we develop a theoretical model based on time-resolved anisotropy to quantify the FRET efficiency of the probes. Additionally, we investigate the conformational dynamics and rotational diffusion of the probes using time-resolved fluorescence anisotropy in homogeneous and heterogeneous environments. Here, we used Ficoll-70 as a heterogeneous crowder to investigate the excluded volume effects on the probes. Measurements in glycerol-enriched buffer were also conducted to distinguish between viscosity and excluded volume effects. Our results indicate that time-resolved anisotropy can be combined with these novel FRET probes for quantitative, non-invasive analysis of site-specific crowding.