Chemistry Reference
In-Depth Information
S
S
+
H
2
N
+
H
2
N
N
+
NH
2
O
N
O
FF
S
N
H
NH
H
2
N
-
O
O
-
S
N
+
+
H
2
N
B
N
+
H
2
N
NH
2
di(ArgCys)-tagged protein
FF
H
NH
O
O
H
2
N
O
-
Orange uorescence
-
O
Green uorescence
fIgure 2.7
Protein fluorescent labelling via di(ArgCys) tag using bodIPy.
protein imaging in live cells has been greatly facilitated using FlAsH and other later-developed bisarsenical fluorophores,
such as the red-fluorescent ReAsH [293-298], especially when protein labelling is prohibited by the large size of fluorescent
proteins [299]. However, concerns should also be kept in mind while using the biarsenical fluorophores, such as background
noise caused by nonspecific binding to endogenous cysteine groups and potential arsenical toxicity.
Fluorette or Dye-Binding Peptide Tag
using phage display, nolan and co-workers developed a set of fluorophore-binding
peptides (fluorettes) for Texas Red with high affinity (binding constants in the picomolar range) [300, 301]. The peptide
aptamers with stably folded structures interact with overlapping regions of Texas Red without interference with the fluores-
cence of Texas Red. one of them could bind to x-rhod calcium sensors, which are structurally similar to Texas Red. Fusion
proteins containing this peptide could bind to the x-rhod calcium sensors in living cells.
Peptides or proteins containing a tetraserine motif (SSPgSS) can bind to a fluorogenic rhodamine-derived bis-boronic
acid with high affinity (in sub-micromolar range), and the binding to the tetraserine-containing peptide sequence was over
four orders of magnitude higher than that with a simple monosaccharide. Imaging of endogenous proteins containing the
SSPgSS motif was attempted in living cells, and fluorescence was turned on only at the SSPgSS-rich cell interior [302].
Recently, a peptide tag containing two pairs of Arg-Cys was designed to label protein with a bodIPy dye covalently via
Michael addition in live cells (Figure 2.7) [303]. This strategy is comparable to the tetracysteine-biarsenic approach, whilst
exhibiting large spectral change and low cytotoxicity.
Metal Ion-Binding Peptide Tag
Metal ion-binding peptide sequences, such as oligohistidine (His-tags) [304] and lantha-
nide-binding tags [305], have been widely used in protein purification, immobilisation, as well as detection [306]. Inspired
by these
in vitro
applications, recent years have seen many examples of metal ion-binding peptide tags in imaging of proteins
in the context of cells [307-311]. For instance, proteins (ligand-gated ion channel and g protein-coupled receptor) fused with
polyhistidine were visualised within seconds in living cells by addition of a chromophore containing a metal-ion-chelating
nitrilotriacetate (nTA) moiety [307].
Biotin Ligase and Lipoic Acid Ligase Peptide Substrate Tags
Ting and co-workers reported the introduction of a bioor-
thogonal tag to cell surface proteins using an approach combining genetic and enzymatic methods (Figure 2.8). First, a
peptide sequence, an
E. coli
biotin ligase (birA) substrate [312], is fused to the target protein genetically, then a biotin
[313, 314] or biotin-mimicking molecule [315], in which two amide nitrogens of biotin are replaced with carbons to pro-
duce a ketone moiety, is introduced to the fusion protein via biotin ligase. The biotin- or ketone-possessing biotin-mimicry
on the protein can then be labelled with fluorescent probes via streptavidin binding or oxime/hydrazine ligation. besides
direct protein staining, the biotin ligase-mediated site-specific labelling was also used to detect protein-protein interactions
in vitro
and in cells, when proteins of interest were fused to birA and birA's peptide substrate, respectively [316]. Although
this proximity biotinylation for protein-protein interaction studies has its advantages, such as high signal to background
ratio and sensitivity, the introduction of birA may alter the protein's behaviour while interacting with other proteins.
In an analogous approach, the Ting lab explored the applicability of lipoic acid ligase (LplA) in place of biotin ligase
(Figure 2.8) [317, 318]. LplA could catalyse the reaction between lipoic acid or its azidoalkyl carboxylic acid analogue (dis-
covered through screening) and the lysine side chain of ligase peptide substrate to form an amide bond. The azido group can
therefore be introduced to protein site-specifically when the protein is fused to LplA's peptide substrate. The Ting lab
