Dynamics of Conformational Transitions in DsRed as Detected by Polarization-Modulated MFICS

Abstract

Non-invasive spectroscopic approaches that are sensitive to fluctuations on the single molecule scale are desirable as methods to characterize the behaviour of proteins in living cells. Here we present molecular Fourier imaging correlation spectroscopy (MFICS) as a novel, high signal-to-noise approach to detect the time-varying translational movement and conformational transitions of the fluorescent protein DsRed. Thermally induced conformational transitions of the DsRed tetramer lead to changes in the optical dipolar coupling between adjacent chromophore sites, and our implementation of polarization- and intensity-modulated photo-excitation generates a phase sensitive signal that is separable into a component that comes from internal, thermally induced conformational transitions and a component which stems from translational diffusion. Based upon joint probability distributions and two-dimensional spectral densities of these measurements in conjunction with existing structural information, we interpret our measurement in terms of transitions between distinct energy-transfer-coupled conformations of DsRed.