Technique / Proteomics and protein biochemistry / Protein-protein interactions

Methods, protocols and troubleshooting on protein protein interactions mapping using FRET, far western blotting and yeast two-hybrid techniques.

Bioluminescence resonance energy transfer (BRET) allows monitoring of protein-protein interactions in real time in living cells. One candidate interacting protein is fused to a luminescent energy donor, such as Renilla luciferase, and the other to a fluorescent energy acceptor, such the green fluorescent protein (GFP), and the two are then coexpressed in the same cells. If the two proteins interact, their close proximity allows nonradiative energy transfer (BRET) between the luciferase and the GFP. BRET does not occur if the two proteins are separated by more than 100 A, making the technique ideal for monitoring protein-protein interactions in biological systems. This unit describes the use of BRET to study constitutive and agonist-promoted interactions among signaling molecules, as illustrated by the homodimerization of the CXCR4 receptor and the recruitment of beta-arrestin2 to agonist-activated G-protein-coupled receptors. This noninvasive and homogeneous assay provides a robust and sensitive proteomic platform with applications for basic science research and drug discovery.

The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant k(OFF) ranged over an order of magnitude from 0.009+/-0.001/s for talin to 0.102+/-0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.