Reversible site-selective labeling of membrane proteins in live cells

Abstract

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.

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

“…Recent progress in protein tagging has allowed us to use different techniques to label proteins in vivo 35,36 . Such techniques will enable us to minimize the sizes of the tags and also potentially overcome the distance problem, because a tag can potentially be placed in proximity to the glycosylation site.…”

A hexahistidine-Zn 2+ -dye label reveals STIM1 surface exposure

“…Although these long-term pathologies need not affect short-term experiments on cultured cells, we see no advantages for Ni 2ϩ . Previous His 6 -binding dyes used on cells (5,6) HisZiFiT is chemically orthogonal and functionally complementary to tetracysteine/biarsenical labeling (Fig. 3).…”

“…Therefore, several groups have devised hybrid genetic/organic alternatives in which a short (Ͻ20 aa), genetically encoded peptide motif binds a small-molecule spectroscopic reporter with sufficient specificity and affinity to be useful in or on living cells (2). Two of the best-known examples are the tetracysteine/biarsenical (1,3) and the hexahistidine/Ni 2ϩ -nitrilotriacetate (His 6 -Ni 2ϩ -NTA) systems (4)(5)(6)(7)(8), which have their own limitations. Tetracysteines function only when fully reduced and are therefore most applicable to the cytosolic and nuclear compartments.…”

Chemical tags: Applications in live cell fluorescence imaging

“…Both reported probes, a NTA-chromophore conjugate [39] and a zinc-chelating fluorophore called HisZiFit [40], show moderate binding affinity to the poly-His peptide (NTA I with K d $ 2 mM; HisZiFit-Zn 2þ with K d $ 40 nM). Weak binding and poor dye characteristics limit the poly-His tag approach to labeling on the cell surface.…”