reproduced verbatim): Protein-protein interaction is a key method by which biological events are regulated. We have developed a new method for measuring protein interactions that overcomes limitations of previous assays. This method, which we call Bioluminescence Resonance Energy Transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and an acceptor fluorophore fused to another protein of interest. If the two candidate proteins interact so as to bring the luciferase and fluorophore in proximity, resonance energy transfer can occur. This interaction is measure as a shift in the color of the bioluminescence emission. BRET will be particularly useful for testing protein interactions within the native cells, especially with integral membrane proteins or proteins targeted to specific organelles. We will apply this method to our topic of primary research interest, """"""""biological clocks,"""""""" and to general applications for the determination of protein-protein interactions within cells and organelles. Biological clocks are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, """"""""jet lag,"""""""" drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, clues to the nature of the underlying biochemical mechanism are only just beginning to emerge. Recent investigations report that protein interactions play key roles in circadian clock mechanisms in eukaryotes. Using well-characterized interacting proteins and proteins encoded by clock genes, we will use the BRET system to 1) test whether results obtained by previous methods for assessing protein interactions can be confirmed with the BRET method, and 2) assay clock protein interactions in situ over the daily cycle to appraise temporal control of protein interaction.
| Borrok, M Jack; Zhu, Yimin; Forest, Katrina T et al. (2009) Structure-based design of a periplasmic binding protein antagonist that prevents domain closure. ACS Chem Biol 4:447-56 |
