| 1. |
- Kalinin, Stanislav, et al.
(författare)
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On the Origin of Broadening of Single-Molecule FRET Efficiency Distributions beyond Shot Noise Limits
- 2010
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:18, s. 6197-6206
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Tidskriftsartikel (refereegranskat)abstract
- Single-molecule FRET experiments on freely diffusing rigid molecules frequently show FRET efficiency (E) distributions broader than those defined by photon statistics. It is often unclear whether the observed extra broadening can be attributed to a physical donor-acceptor distance (R-DA) distribution. Using double-stranded DNA (dsDNA) labeled with Alexa488 and Cy5 (or Alexa647) as a test system, we investigate various possible contributions to the E distribution width. On the basis of simultaneous analysis of donor and acceptor intensities and donor lifetimes, we conclude that dsDNA chain dynamics can be ruled out as a possible reason for the observed E distribution broadening. We applied a set of tools to demonstrate that complex acceptor dye photophysics can represent a major contribution to the E distribution width. Quantitative analysis of the correlation between FRET efficiency and donor fluorescence lifetime in 2D multiparameter histograms allows one to distinguish between broadening due to distinct FRET or dye species. Moreover, we derived a simple theory, which predicts that the apparent distance width due to acceptor fluorescence quantum yield variations increases linearly with physical donor-acceptor distance. This theory nicely explains the experimentally observed FRET broadening of a series of freely diffusing labeled dsDNA and dsRNA molecules. Accounting for multiple acceptor states allowed the fitting of experimental E distributions, assuming a single fixed donor-acceptor distance.
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| 2. |
- Sisamakis, Evangelos, et al.
(författare)
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ACCURATE SINGLE-MOLECULE FRET STUDIES USING MULTIPARAMETER FLUORESCENCE DETECTION : SINGLE MOLECULE TOOLS, PT B
- 2010
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 474, s. 455-514
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Forskningsöversikt (refereegranskat)abstract
- In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Forster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of sm FRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules. After a short introduction, the data analysis procedure is described in detail together with some experimental considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy. Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its structural energy landscape.
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