Chemistry Reference
In-Depth Information
variety of different
fluorophores in live mammalian cells and the approach can be
used for multicolor analysis of cellular processes and FRETmeasurements [27, 28].
A mutant form of AGT was recently created that can be speci
cally labeled
with substrates that are not accepted by wild-type AGT [29]. This permits the labeling
of two different AGT fusion proteins with various
fluorophores in the same cell or
in vitro.
7.2.4
Acyl-carrier Protein (ACP)
This labeling comprises the enzymatic transfer (using speci
c phosphopanteinyl
transferases) of the phosphopantetheine of a
uorescent Coenzyme A (CoA)
conjugate to an ACP fused to the target protein [30
-
32]. This technique enabled
us to track single G protein-coupled receptors (GPCRs) during signaling because
several obstacles had been overcome: (i) proteins not properly inserted in the plasma
membrane were not labeled and hence did not contribute to out-of-focus background
fluorescence. (ii) The fraction of labeled protein could be precisely controlled in time
(pulse labeling) with the possibility of labeling repetitively at subsequent times,
ensuring a low and well de
ned dye concentration required for single-molecule
detection. (iii) Free choice of probes allows protein labeling with long-wavelength
dyes (for example Cy5, Atto dyes) of high absorption cross section, high quantum
yield and high photo-stability to be used as label for an improved signal-to-
background ratio and observation time. (iv) Multicolor labeling of de
ned ratio(s)
between different probes is easily possible by controlling the composition of
substrates in the bulk medium. (v) Non-speci
c binding of the label to the plasma
membrane, the most crucial obstacle for the successful application of single-
molecule microscopies, was reduced by the presence of the hydrophilic CoA-moiety.
(vi) Recent developments reduced the size of the ACP tag from 76 to 11 residues
without losing too much speci
city and ef
ciency of the labeling reactions, making
this approach even more attractive [33].
7.2.5
Nitrilotriacetate (NTA)
This is a generic method for selectively labeling proteins in vivo and in vitro, rapidly
(within seconds) and reversibly, with small molecular probes that can have a wide
variety of properties [34]. The probes comprise a chromophore and a metal ion-
chelating NTA moiety, which binds reversibly and speci
cally to engineered oligo-
histidine sequences in proteins of interest. The feasibility of the approach was
demonstrated by binding NTA
-
chromophore conjugates to a representative ligand-
gated ion channel and a GPCR, each containing a polyhistidine sequence. In contrast
to the transient binding of such a conventional mono-NTA-bearing
uorophore,
multivalent-NTA-bearing
fluorophores form complexes with oligohistidine se-
quences in proteins, which show increased lifetimes of more than 1 h [35, 36].
