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. 2008 Aug;18(4):516-24.
doi: 10.1016/j.sbi.2008.06.008. Epub 2008 Aug 12.

Fluorescence characterization of denatured proteins

Huimin Chen  1 , Elizabeth Rhoades
Affiliations

Fluorescence characterization of denatured proteins

Huimin Chen et al. Curr Opin Struct Biol. 2008 Aug.

Abstract

Characterization of unfolded states, while critical to a complete understanding of protein folding, is inherently difficult due to structural heterogeneity and dynamic interchange between states. The growing body of work focusing on single molecule fluorescence techniques for the study of protein folding, also highlights their potential for studies of unfolded proteins. These methods can obtain conformational information about individual subpopulations of molecules in an ensemble, and measure dynamics without the need for synchronization. The studies highlighted here demonstrate the promise of these techniques for obtaining novel information about unfolded states in vitro and in more physiologically relevant milieu.

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Figures

Figure 1
Figure 1
Schematic of typical setup for smFRET or FCS. A laser, most commonly CW (although pulsed lasers which allow for TCSPC measurements have being used in a number of recent studies [27,30,36] is focused by a high NA (>0.9) objective lens to a diffraction-limited focal volume (~10−15 liter). Emitted fluorescence is collected through the objective, separated from the excitation beam by a dichroic mirror and focused onto a confocal aperture that provides depth discrimination. Donor and acceptor signals are separated by dichroic mirrors in smFRET or by a beamsplitter for cross-correlation of the signal in FCS before detection. (A) Measurements of freely diffusing molecules (pM for smFRET and nM for FCS) results in ‘bursts’ of photons which are further processed, as described in Box 2 and Box 3. (B) A single immobilized molecule must be first detected and localized in the focal volume to allow for measurement of an intensity trace, which typically lasts for a few seconds before photobleaching. A total internal reflection (TIR) setup, not shown here, allows for simultaneous measurement of intensity traces from multiple immobilized molecules albeit with reduced time resolution (see [7] review for further discussion).
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References

    1. Bilsel O, Matthews CR. Molecular dimensions and their distributions in early folding intermediates. Curr Opin Struct Biol. 2006;16:86–93. - PubMed
    1. Haas E. The study of protein folding and dynamics by determination of intramolecular distance distributions and their fluctuations using ensemble and single-molecule FRET measurements. ChemPhysChem. 2005;6:858–870. - PubMed
    1. Kubelka J, Hofrichter J, Eaton WA. The protein folding 'speed limit'. Curr Opin Struct Biol. 2004;14:76–88. - PubMed
    1. Mittag T, Forman-Kay JD. Atomic-level characterization of disordered protein ensembles. Curr Opin Struct Biol. 2007;17:3–14. - PubMed
    1. Stryer L, Haugland RP. Energy Transfer - a Spectroscopic Ruler. Proc Natl Acad Sci U S A. 1967;58:719-&. - PMC - PubMed

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