On the control of TCR phosphorylation
Fernandes RA, Huo J, Lui Y, Felce JH, Davis SJ. (2012), Front Immunol. 3, 92
Key figure: Analysis of protein interactions using bioluminescence resonance energy transfer (BRET)
(A) Schematic showing a monomeric protein expressed at the cell surface as chimeras with luciferase (Luc) and a green fluorescent protein (GFP) acting as donors and acceptors, respectively. The blue circle represents the 100 Å sphere within which random or stable co-association of Luc and GFP allows energy transfer. (B) Graphical representation of the relationship of energy transfer efficiency (BRETeff) to the acceptor/donor ratio for dimeric (green) and monomeric (blue) protein pairs expressed at the membrane, versus that measured for a pair of proteins one of which is expressed at the membrane (mem) and the other in the cytoplasm (cyt; James et al., 2006, 2011). In practice, the transfer efficiency is normalized against that measured for a soluble GFP/Luc fusion protein (sGFP–Luc) expressed in the cytoplasm. (C) The efficiency of energy transfer arising viarandom interactions is explained by the high density of membrane surface proteins and by their high mobility. Protein density is illustrated to scale, assuming that there are 20,000 molecules/μm2, and that each protein is 4 nm in diameter (Grasberger et al., 1986). It takes 0.2–0.3 s for a protein to move from position A to position B, based on measurements of the TCR diffusion rate (James et al., 2007). Since many of the proteins are likely diffusing at comparable rates, numerous random interactions seem unavoidable. Some estimates for the expression levels of cell surface proteins are substantially higher (Quinn et al., 1984).