Comparative Protein Dynamics with Droids 1.0 - A Gui-Based Pipeline for Functional Evolutionary Protein Analysis and Visualization

Abstract

describe the rotational dynamics of hetero-FRET biosensors using timeresolved anisotropy. The model was tested on wavelength-dependent experiments of hetero-FRET probes (mCerulean-linker-mCitrine) with linkers of varying length and flexibility in buffer at room temperature. This model guided our experimental design towards the optimum wavelengthdependent time-resolved anisotropy that provides the most sensitivity to macromolecular crowding. Using this model to fit our experimental results, we are able to determine the energy transfer rate constant and the energy transfer efficiency of these hetero-FRET biosensors. Our findings are in general agreement with the standard fluorescence lifetime-based FRET analysis. However, wavelength-dependence of time-resolved anisotropy and its sensitivity to the energy transfer efficiency are rather distinct from time-resolved fluorescence approaches. Currently, we are using the developed model to investigate the energy transfer efficiency of selected FRET sensors in homogeneous and heterogeneous environments. Our results represent an important step towards the application of quantitative and non-invasive time-resolved anisotropy of hetero-FRET probes to investigate macromolecular crowding and protein-protein interactions in living cells.