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Determination of the Orientational Distribution and Orientation Factor for Transfer between Membrane-Bound Fluorophores using a Confocal Microscope

Ben Corry ^*, Dylan Jayatilaka ^*, Boris Martinac  and Paul Rigby 

^* School of Biomedical, Biomolecular and Chemical Sciences, School of Medicine and Pharmacology, and Biomedical Imaging and Analysis Facility, The University of Western Australia, Crawley, Australia

Correspondence: Address reprint requests to B. Corry, E-mail: ben{at}theochem.uwa.edu.au.

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound fluorophores. We demonstrate how information about the orientation of fluorophores can be used to calculate the orientation factor ² required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.

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