Separating Structural Heterogeneities from Stochastic Variations in Fluorescence Resonance Energy Transfer Distributions via Photon Distribution Analysis
- 15 March 2006
- journal article
- research article
- Published by American Chemical Society (ACS) in The Journal of Physical Chemistry B
- Vol. 110 (13), 6970-6978
- https://doi.org/10.1021/jp057257+
Abstract
We establish a probability distribution analysis (PDA) method for the analysis of fluorescence resonance energy transfer (FRET) signals to determine with high precision the originating value of a shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background and represent the minimum width that a FRET distribution must have. In this way an unambiguous distinction is made between shot-noise distributions and distributions broadened by heterogeneities. This method simultaneously and effectively extracts highly resolved information from FRET distributions. The theoretical histograms match the exact profile of histograms generated from constant transfer efficiency experimental data with a χ2 near unity. The χ2 surface suggests an ultimate level of precision with FRET of <1% of the Förster radius. Distributions of FRET signals in donor−acceptor-labeled DNA were unambiguously identified as being broader than shot-noise variations could explain. A model describing a Gaussian distribution of distances was tested with the PDA method and demonstrated 5 Å inhomogeneities due to dye motions. The capability of this method to recover quantitative information from FRET distributions has potential applications for studying molecular conformations and dynamics. Potential sources for artifacts such as acceptor photobleaching, spectrally different observation volumes, and fluctuations of the Förster radius are ruled out.Keywords
This publication has 23 references indexed in Scilit:
- Analysis of Photobleaching in Single-Molecule Multicolor Excitation and Förster Resonance Energy Transfer MeasurementsThe Journal of Physical Chemistry A, 2006
- The Study of Protein Folding and Dynamics by Determination of Intramolecular Distance Distributions and Their Fluctuations Using Ensemble and Single‐Molecule FRET MeasurementsChemphyschem, 2005
- Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical SwitchPhysical Review Letters, 2005
- Carbocyanine Dyes as Efficient Reversible Single-Molecule Optical SwitchJournal of the American Chemical Society, 2005
- Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthaseNature Structural & Molecular Biology, 2004
- Photodestruction Intermediates Probed by an Adjacent Reporter MoleculePhysical Review Letters, 2003
- Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopyNature, 2002
- Two New Concepts to Measure Fluorescence Resonance Energy Transfer via Fluorescence Correlation Spectroscopy: Theory and Experimental RealizationsThe Journal of Physical Chemistry A, 2001
- Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation SpectroscopyThe Journal of Physical Chemistry A, 2000
- Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence SpectroscopyThe Journal of Physical Chemistry A, 1998