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...

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Bibliographic Details
Published in:The journal of physical chemistry. B 2009-05, Vol.113 (19), p.6847-6853
Main Authors: Lott, Geoffrey A, Senning, Eric N, Fink, Michael C, Marcus, Andrew H
Format: Article
Language:English
Subjects:
Animals
Anthozoa
B: Biophysical Chemistry
Biopolymers - chemistry
Fourier Analysis
Luminescent Proteins - chemistry
Molecular Conformation
Spectrum Analysis
Time Factors
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Summary: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 ∼ 106). 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.
ISSN:1520-6106
1520-5207
DOI:10.1021/jp8093116