Accurate FRET Measurements Resolving Distances and Dynamics in Biomolecules

Abstract

Fluorescence spectroscopy and imaging are important biophysical techniques to study dynamics and function of biomolecules in vitro and in live cells. The use of more than one fluorophore per molecule opens additional opportunities arising from photon densities, coincidences and dipolar coupling by Förster Resonance Energy Transfer (FRET) to study the stoichiometry and structure of biomolecular systems. Before performing FRET measurements, one needs to know four calibration parameters, which depends on the photophysical properties of the used dyes and characteristics of optical components. We used a rational determination to estimate the calibration factors using DNA samples and compared this to a calibration free lifetime based analysis. This results in resolving individual γ-factors for every different set of dye pairs in different physical environments enabling an accurate monitoring of the operating dyes. Comparison and error estimation to the analysis with global parameters shows differences for specific dye pairs. Using this approach a network of FRET pairs in combination with integrative modeling is able to resolve the structure and dynamics of a biomolecule like the hGBP1. The shown extended network with more dye pairs results in a higher resolution of the structural model. To analyze the dynamic behavior of the biomolecule different several techniques like Multiparameter Fluorescence Detection (MFD), Photon Distribution Analysis (PDA) and filtered Fluorescence Correlation Spectroscopy (fFCS) are used. This workflow opens insights into several different dynamic proteins.