I. Conformational dynamics of biological macromolecules by polarization-modulated Fourier imaging correlation spectroscopy.
Experiments that optically probe the translational motions and internal conformational transitions of biological macromolecules have the potential to enable mechanistic studies of biochemical processes in living cells. This work presents a novel "phase-selective" approach to fluorescence fluctuation spectroscopy that simultaneously monitors protein conformational transitions and nanometer center-of-mass displacements. Polarization- and intensity-modulated photoexcitation is combined with phase-sensitive signal detection to monitor the collective coordinate fluctuations from a large population of fluorescent molecules (N approximately 10(6)). Test experiments are performed on DsRed, a tetrameric complex of fluorescent protein subunits. Thermally induced conformational transitions of the complex lead to fluctuations in the optical dipolar coupling between adjacent chromophore sites. Polarization-resolved equilibrium fluctuation trajectories provide the raw data necessary to determine time-correlation functions and probability distributions of coordinate displacements, which characterize conformational transitions of the DsRed complex.
Lott
Geoffrey A
GA
Department of Physics, University of Oregon, Eugene, Oregon 97403, USA.
Senning
Eric N
EN
Fink
Michael C
MC
Marcus
Andrew H
AH
D000818:Animals; D037421:Anthozoa; D001704:Biopolymers / Q000737:chemistry*; D005583:Fourier Analysis; D008164:Luminescent Proteins / Q000737:chemistry; D008968:Molecular Conformation; D013057:Spectrum Analysis; D013997:Time Factors
D001704:Biopolymers; D008164:Luminescent Proteins; C413663:fluorescent protein 583