Green fluorescent protein , FRET

Squire, A., Verveer, P. J., Pocks, O. and Bastiaens, P. I. H. (2004). Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells. J. Struct. Biol. 147, 62-9. 

Ruiz-Velasco, V. and Ikeda, S. R. (2001). Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons. J. Physiol. 537, 679-92. 

Features a new box on visualizing biochemistry with fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP)... 

Fig. 7. Topological modifications to increase a FRET signal between two variants of the green fluorescent protein (GFP) fused to interacting proteins, (a) Schematic representation of interacting proteins and GFP variants with the location of the N- and C-termini indicated, (b) The GFP variants fused to the ends of the interacting proteins are too far apart to generate a FRET signal, (c) Insertion of one of the GFP variants into an interacting protein brings the two GFP variants close enough for FRET to be detected.

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

Like FRET, bioluminescence resonance energy transfer (BRET) is based on non-radiative energy transfer between a donor and an acceptor. However, in BRET, the donor is a luminescent molecule, excited by the enzyme Renilla Luciferase (Rluc), and not a fluorescent molecule. The BRET acceptor can be a fluorescent protein like green fluorescent protein (GFP) or YFR If the enzyme and the fluorescent protein are in close proximity (d < 10 nm), an energy transfer will occur between the Rluc substrate (coelanterazine) and the fluorescent protein, leading to emission from the later. 

Volkmer A, Subramaniam V, Birch DJS, Jovin TM. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys. J. 2000 78 1589-1598. Subramaniam V, Hanley QS, Clayton AHA, Jovin TM. Photophysics of green and red fluorescent proteins implications for quantitative microscopy. Methods Enzymol. 2003 360 178-201. Rizzo MA, Springer GH, Granada B, Fdston DW. An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnol. 2004 22 445-449. 

Abbreviations Acs, acetyl coenzyme A synthetase CheA P, phosphorylated CheA CheY P, phosphorylated CheY FRET, fluorescence resonance energy transfer GFP, green-fluorescent protein PTS, phosphoenolpyruvate-dependent carbohydrate phosphotransferase system YFP, yellow-fluorescent protein. 

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