Chemistry Reference
In-Depth Information
Several strategies have been developed in recent years to overcome some of the
limitations within the methods mentioned above. A few recent reviews summarize
these efforts [19
-
21]. In the following we report on our efforts to use the novel
approaches for single-molecule microscopies.
7.2.2
Suppressor tRNA Technology
Several methods have been developed to incorporate unnatural amino acids
site-speci
cally into proteins in mammalian cells. Chemically aminoacylated
suppressor tRNAs have been microinjected or electroporated into CHO cells and
neurons, and used to suppress nonsense amber mutations with a series of unnatural
amino acids [22]. Thereby it was possible to incorporate unnatural amino acids
with diverse physicochemical and biological properties into de
ned positions of
the sequence of target proteins expressed in mammalian cells. This method
has been widely used to probe channel proteins by electrophysiology [23] and was
used for the
first time by Turcatti et al. [24] to insert non-natural
uorescent
amino acids at speci
c sites in a GPCR (NK2 receptor) in frog oocytes for
exploiting by
fluorescence resonance energy transfer (FRET) the receptor
structure. Meanwhile, Schultz et al. extended the method in an elegant, general
approach (for a recent review see [25]): a mutant Escherichia coli aminoacyl-tRNA
synthetase (aaRS) is
rst evolved in yeast to selectively aminoacylate its tRNAwith the
unnatural amino acid of interest. This mutant aaRS together with an amber
suppressor tRNA is then used to site-speci
cally incorporate the unnatural amino
acid into a protein in mammalian cells in response to an amber nonsense codon.
This and other approaches [26] overcome the originally low ef
ciency of expressed
proteins.
7.2.3
O6-Alkylguanine
-
DNA Alkyltransferase (AGT)
The labeling is based on the irreversible and speci
c reaction of human
O6-alkylguanine
-
DNA alkyltransferase (hAGT) with
fluorescent derivatives of O
6
-
benzylguanine (BG), leading to the transfer of the synthetic probe to a reactive
cysteine residue on hAGT which is fused to a protein of interest [27, 28].
Wild-type hAGT is a monomeric protein of 207 aa, the 30 C-terminal residues
of which can be deleted without affecting the reactivity against BG, making it
smaller than auto
uorescent proteins. Importantly, the rate of the reaction of AGT
fusion proteins with BG derivatives is independent of the nature of the label, opening
up the possibility of labeling a single AGT fusion protein with a variety of
different probes. Speci
c labeling of AGT fusion proteins in mammalian cells can
be achieved by using AGT-de
cient cell lines, and it has been shown that nuclear-
localized AGTcan be labeled speci
fluorescein in such cells [27]. Synthetic
BG derivatives can be used for the sequential labeling of AGT fusion proteins with a
cally with
