Reversible site-selective labeling of membrane proteins in live cells.
Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each containing a polyhistidine sequence. We investigated the ionotropic 5HT(3) serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.
Guignet
Emmanuel G
EG
Hovius
Ruud
R
Vogel
Horst
H
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007477:Ions; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011485:Protein Binding; D013997:Time Factors
D007477:Ions; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D009571:Nitrilotriacetic Acid