With chemical tags, rather than tagging the target protein with an FP, the protein is tagged with a polypeptide, which is subsequently modified with an organic fluorophore. Technically, labeling a target protein with a chemical tag is very similar to labeling with FPs: a plasmid encoding a fusion between the target protein and polypeptide tag is constructed using standard molecular cloning techniques and then introduced into the desired cell. The transfected cells are briefly incubated with the organic fluorophore, which diffuses into cells and is then specifically bound by or conjugated to the polypeptide tag. Thus, chemical tags retain the specificity of protein labeling achieved with FPs through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities.
The first report of a chemical surrogate to FPs for labeling proteins with organic fluorophores in living cells was FlAsH from Tsien and co-workers in 1998.9
In design, FlAsH is the ideal chemical tag—a short 15 amino acid polypeptide tag with a tetracysteine core (CCXXCC) that is covalently labeled with a fluorogenic bisarsenical fluorescein ligand whose fluorescence increases upon binding to the polypeptide tag (). To date, a number of bisarsenical fluorophores and corresponding tetracysteine (TC) tags have been reported,10–12
among which the original green-fluorescent FlAsH and the red-fluorescent ReAsH are most frequently utilized. Despite its elegant design, the FlAsH technology suffers practically from non-specific labeling of thiol-rich biomolecules in the cell and toxicity of the bisarsenical ligands and labeling conditions.13
Nonetheless, benefiting from its small size, FlAsH often is the only viable tag for labeling proteins or complexes impaired by the ~ 250 amino acid FPs and is widely reported and has enabled many experiments not otherwise possible.14
Figure 1 Schematic illustration of representative example technologies of different strategies for selectively labeling proteins in living cells. (A) The FlAsH tag features a short peptide with a tetracysteine core that directly binds bisarsenical fluorogens. (more ...)
Protein-based chemical tags
To overcome the selectivity limitations of a short peptide tag, protein-based chemical tags were introduced that allowed the target protein to be tagged with a protein receptor or enzyme, which can be subsequently labeled with a cell-permeable organic ligand- or substrate-probe heterodimer. There are several critical design issues for these protein receptor-ligand or enzyme-substrate tags. One, and perhaps most importantly, the organic fluorophore ligand or substrate must be readily cell-permeable and not binding non-specifically to endogenous proteins or other biomolecules or, equally important but perhaps less appreciated, otherwise partitioning to particular organelles within the cell. Two, the synthesis of the ligand or substrate derivatives should be straightforward and minimally disruptive to the receptor binding or enzyme function. Three, the protein receptor or enzyme should be small, monomeric, and well behaved for minimal perturbation of the biological pathway being studied. Fourth, the labeling reaction between the protein tag and the ligand/substrate-probe should be rapid and near quantitative. To date, the advantage of protein tags over other chemical tags is that they are sufficiently selective and efficient to enable intracellular proteins to be imaged with high signal-to-noise.
In collaboration with the Sheetz group, our laboratory has exploited the high-affinity interaction between dihydrofolate reductase (DHFR) and folate analogs to label proteins in living cells. Briefly, we tag the target protein with E. coli
DHFR (eDHFR). Because eDHFR binds trimpethoprim (TMP) with high affinity (1 nM KD
) and selectivity (affinities for mammalian DHFRs are KD
> 1 μM), the eDHFR tag can then be labeled with near stoichiometric concentrations of TMP-probe heterodimers that bind to eDHFR non-covalently with a dissociation half-life of tens of minutes ().19,30,31
Consistent with TMP's use therapeutically as an antibiotic, the TMP-probe heterodimers have excellent cell permeability and solubility properties. As anticipated based on high-resolution structural and SAR data, commercially available TMP can be modified at the para-methoxy position with only minor perturbation of high-affinity binding to eDHFR, making the synthesis of TMP-probe labels very straightforward. As a 159 amino acid, monomeric, well-behaved protein, eDHFR is about two-thirds the size of FPs, does not suffer from oligomerization and expression problems, folds rapidly, and circumvents the issue of chromophore maturation half-life.32,33
Among chemical tags, the TMP-tag stands out for enabling the imaging of intracellular
proteins with high resolution in living cells.
Our efforts to image individual proteins in the focal adhesion complex in mammalian cells using the TMP-tag taught us that the key performance issue for the tag is not so much the cell-permeability of the TMP-probe label, but really the solubility of the TMP-probe label once it is inside the cell. Significantly, through optimization of protecting groups and linkers, we obtained TMP-fluorophore labels that exhibited minimal non-specific partitioning to lipid-rich cellular compartments and could thus be utilized to image more dilute, rapidly diffusing cytoplasmic proteins with high signal-to-noise.19
The palette of TMP-fluorophores able to image intracellular proteins has been expanded from fluorescein-based green and red dyes to include a far-red photo-switching Atto-655, a two-photon fluorophore BC575, and lanthanide probes.20,34,35
Important for adoption by cell biologists and biophysicists (i.e. laboratories not specializing in organic synthesis), the TMP-tag is commercially available from Active Motif as LigandLink™.
Remarkably, the TMP-tag now has also been rendered covalent by installing a unique Cys residue on eDHFR in position to react with a latent acrylamide electrophile added to the TMP-probe molecule via a classic proximity-induced Michael addition.36
While under optimization, already this first-generation covalent TMP-tag undergoes rapid, quatitative covalent labeling (in vitro
~ 50 min) and enables imaging of nuclear-localized eDHFR in live NIH3T3 cells. This work demonstrates that an advantage to a chemical tag based on high-affinity binding is that it does not require the high concentration of ligand-probe conjugate necessary with enzyme-based chemical tags, where KM
s typically range from μM to mM, leading to high background noise from unbound fluorophore and necessitating extensive washing steps.29,37
An alternative strategy to protein-based chemical tags, the “SNAP-tag” utilizes O6
-modified guanine-fluorophore heterodimers to covalently label proteins fused to human O6
-alkylguanine-DNA-alkyltransferase (hAGT), a 20kD, monomeric DNA repair protein that naturally dealkylates O6
-alkylated guanine residues in damaged DNA by a single turnover alkylation of an active-site Cys residue ().37
A fast-reacting SNAP-tag variant has been engineered to minimize background labeling of endogenous mammalian AGT.38
Impressively, an orthogonal AGT variant that selectively uses cytosine-fluorophores as substrates, called CLIP-tag, has been evolved, although it will require further optimization to be as robust as the SNAP-tag.37,39
A wide range of SNAP- and CILP-fluorophores are commercially available from New England Biolabs, and a subset of these have been confirmed to work inside living cells (). Likewise, Promega has developed a covalent chemical tag based on the reaction of an engineered dehalogenase enzyme with a suicide substrate—“HaloTag”,26,28
which has been shown to be a useful tag in vitro
, but reports indicate may suffer efficiency and selectivity issues inside cells.40
Chemical tags and corresponding fluorophores used to image functional proteins inside living mammalian cells
New protein-based chemical tags continue to be introduced, but most of these are yet to be sufficiently vetted, particularly for the demanding task of labeling proteins intracellularly, to judge their practical performance at this time. These tags include a cutinase variant that reacts with a suicide substrate;41
a fluorogenic tag in which a β-lactamase variant displaces a quencher from a cephem suicide substrate;42,43
and a non-covalent tag where synthetic ligand of FK506 (SLF) binds to FKBP12:F36V.44
Peptide-based chemical tags
With chemical tagging, it should be possible to replace protein tags, which can interfere with protein interactions and pathway function, with very short peptide tags. The challenge for the field now is to devise conceptually new strategies for chemically modifying short peptides with fluorophores that overcome current limitations in the selectivity with which a short peptide tag can be recognized over the many other reactive molecules present in a living cell.
The Ting laboratory has pioneered the re-engineering of natural enzymes that modify short peptides with organic molecules to fluorophore labeling.45
The most advanced of these chemical tags, the E. coli
biotin ligase (BirA) enzyme, whose natural function is biotinylation of proteins containing a peptide recognition motif, is used to label a 15 amino acid peptide tag with a biotin analog, which is subsequently modified with a fluorophore ().45,46
To date BirA has resisted more dramatic modification to enable direct incorporation of a biotin-fluorophore conjugate. Intracellular protein labeling, therefore, is still difficult because the second reaction between the biotin analog and the fluorophore is slow and incompletely selective at the μM concentrations typical in the cell. An exciting recent advance for the potential it illustrates, a lipoic acid ligase variant was evolved to use a coumarin derivative directly, although coumarin itself is not an ideal fluorophore for live cell imaging.
A variety of enzyme-mediated peptide tags have now been reported, including the acyl-carrier protein (ACP)-based tag modified by phosphopantetheine transferase (PPTase);47
the 6-amino acid peptide modified by sortase;48,49
and the formylglycine-generating enzyme-based tag reported by Bertozzi.50
To date all of these aforementioned peptide tags have only been demonstrated to work on the cell surface.
Beyond enzyme-mediated peptide tags, other clever approaches to short peptide tags are being explored. Similar to the FlAsH tag, a tetraserine peptide tag was demonstrated to bind a fluorogenic bis-boronic acid derivative.51
In a very recent report, Chang and coworkers developed a peptide tag that undergoes a Michael addition with a BODIPY fluorophore.52
Inspired by the popular polyhistidine tag for protein purification, various metal-chelating peptides have been adapted for protein labeling.53–57
In an interesting approach, Nolan and co-workers evolved a 38-amino acid peptide that binds the Texas red fluorophore with high affinity.58
An N-terminal Cys residue generated in vivo
with a sequence-specific protease has been labeled with thioesters, analogous to native chemical ligation.59
Although these different strategies are at an early stage of development, they illustrate the creativity with which chemistry can be exploited in a living cell, making a significant impact not only for live cell imaging, but more broadly for synthetic biology.