Abstract

Fluorescence Resonance Energy Transfer (FRET) spectroscopy is an ideal tool to assess protein-protein interactions in cells. FRET occurs if suitable fluorophores are less than 10nm apart and thus may be used to probe molecular scale interactions. To obviate the need for fixation and staining, the proteins of interest can be linked to Green Fluorescent Protein (GFP) variants, which can then be expressed in intact living cells. However, FRET is inherently difficult to validate and interpret in such environments, making suitable positive controls vital. In this study cyan and yellow variants of GFP (CFP and YFP) were linked by amino acid chains of known lengths (6, 14, 16, 18, and 22 amino acids). Ratiometric FRET measurements were obtained via the acceptor photobleaching technique. The data was analyzed by new FRET analysis software developed by this group, which will be made available to the public. The evaluation method is novel in that it assesses an entire field of view for FRET efficiencies, thus making high throughput data analysis possible. A perfect candidate for FRET studies is the cyclin-CDK switch, responsible for the regulation of mitosis. Preliminary results with CyclinB1-CFP and cdk1-YFP are presented.

© 2003 SPIE

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