Single pair FRET

Cameleon - single-pair FRET between two GFP mutants in a [95]... 

Probing Protein Conformational Changes by Intramolecular Single-Pair FRET... 

Single pair FRET has also been used to image macromolecular interactions at the single molecule level. FRET takes place only if the donor and acceptor fluorophores are very near to each other (distance, <10 nm). FRET is the most suitable method for detecting the complex formation between two molecules, whereas observation of co-locahzation of two molecules is sometimes difficult to interpret because of the diffraction Hmit. Ishii et aL (1999) measured FRET between single Cy3 and Cy5 fluorophores conjugated separately to a-tropomyosin subunits [14]. When the two subunits of a-tropomyosin attached to the different fluorophores formed a homodimer, the two fluorophores were close enough... 

In this chapter we will briefly discuss the photophysical properties of commonly used dyes and, by reference to biomolecular systems studied by single molecule FRET in the literature, discuss the properties of commonly used dye pairs. We will then describe, in detail, the labelling and purification methods used in our laboratories to generate milligram quantities of a single chain protein labelled with two different dyes. Finally, we will discuss a variety of methods by which biomolecular systems have been tethered at or close to a surface, a necessary prerequisite for performing many of the experiments described in other chapters. 

An important feature that influences the performance of the probe to a large extent is the selection of the FRET pair. Generally, fluorophores with high excitation wavelengths are preferred over blue fluorophores in single photon microscopy for applications in tissue since there is no autofluorescence in this region and the ratio... 

FRET Well described FRET pairs, mostly single donor-single acceptor configurations, enables optimization of reporter properties Few examples, single donor-multiple acceptor configurations possible, limitation of FRET efficiency due to nanometer-size of QD-coating... 

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