Marcia Levitus
Research
Overview Nucleosome Dynamics Photophysics FCS-FRET
Fluorescence Correlation Spectroscopy
Fluorescence Correlation Spectroscopy (FCS) has recently emerged as a powerful technique to study conformational dynamics of samples containing a small number of molecules. This is achieved by quantifying the fluorescence fluctuations of a sample containing a fluorophore-quencher combination covalently attached to strategic positions in the biomolecule of interest. Measurements are performed in solution, where diffusion of the sample in and out of the observation volume creates fluctuations that are measured on top of the fluctuations produced by the conformational dynamics under study. The need to separate the contributions due to kinetics from the contributions due to diffusion represents the main drawback that limits the applicability of this technique
We developed a new approach based on the simultaneous measurement and analysis of the auto- and cross-correlation functions of the intensities measured in two independent detectors for a biomolecule containing a fluorescent donor-acceptor pair. Fluctuations in both the donor and acceptor intensities are observed due to variations in the efficiency of Fluorescence Resonance Energy Transfer (FRET) as the donor-acceptor distance changes due to conformational dynamics. We analyzed a generic case in which a macromolecule exists in equilibrium between two conformations with distinct FRET efficiencies. Although the complete description of the auto- and cross-correlation decays requires that the diffusion characteristics of the system are taken into account, the ratio of any two independent decays depends on kinetic parameters only. Thus, we propose that the simultaneous measurement of the donor-donor, acceptor-acceptor, and donor-acceptor correlation functions provides a means by which kinetic information can be obtained without the need of characterizing the diffusion properties of the system.