Chemical tags for labeling proteins inside living cells

Abstract

To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, we now face the challenge of understanding how macromolecular machines, signaling pathways, and other biological networks operate in the complex environment of the living cell. The fluorescent proteins (FPs) revolutionized our ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biology, they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chemical methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chemical tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chemical tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an organic fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chemical tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, we focus on the development and application of chemical tags for studying protein function within living cells. Thus, in our overview of different chemical tagging strategies and technologies, we emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. We highlight recent applications in which the chemical tags have enabled sophisticated biophysical measurements that would be difficult or even impossible with FPs. Finally, we conclude by looking forward to (i) the development of high-photon-output chemical tags compatible with living cells to enable high-resolution imaging, (ii) the realization of the potential of the chemical tags to significantly reduce tag size, and (iii) the exploitation of the modular chemical tag label to go beyond fluorescent imaging.

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. (B) The eDHFR/TMP labeling strategy is based on non-covalent, high-affinity binding of TMP-probe heterodimers by the protein eDHFR. (C) In the SNAP-Tag, the enzyme hAGT utilizes a guanine-probe heterodimer as a suicide substrate. (D) Biotin ligase enzymatically modifies a short peptide tag with a biotin analogue, which then reacts with the probe in a second step.

Figure 2

Experimental set up and representative single-molecule trace of pre-mRNA splicing in yeast cell extracts imaged with chemical tags, providing a unique way to access the dynamic mechanism of pre-mRNA splicing. (A) Pre-mRNA labeled with Alexa488 is immobilized on a glass slide. Pairs of the snRNP complexes that make up the spliceosome, shown here U1 and U2, are genetically encoded in fusion with eDHFR and hAGT, respectively, allowing for orthogonal labeling with TMP-Cy5 and SNAP-DY549 in yeast cell extract. (B) The arrival and departure of each snRNP complex is visualized as the appearance and disappearance of fluorescence signal above the baseline. The delay between U1 binding and U2 binding provided direct evidence that assembly of the spliceosomal components is a highly ordered process and, surprisingly, the single-molecule traces also showed the association and disassociation of each component is highly dynamic.

Figure 3

Super-resolution dSTORM imaging of histone protein 2B (H2B) using TMP-tag. (A) In conventional confocal microscopy, the resolution set by the diffraction limit of light is ~200 nm, and thus individual protein molecules cannot be resolved. In PALM/STORM, small percentages of the total population of fluorophores are randomly photo-activated over time, allowing all individual fluorophores to be localized to resolutions of ~20 nm from the Gaussian fits of their point spread functions. (B) Total internal reflection fluorescence image of TMP-Atto655 tagged H2B in the nucleus of living HeLa cells and (C) corresponding dSTORM image with improved resolution. The expanded views and cross-sectional profiles (D, E) demonstrate superior resolution well below the diffraction barrier. Adjacent histone proteins separated by ~ 100 nm are clearly resolved.

Figure 4

Pulse-chase labeling of C×43 with FlAsH and ReAsH and correlative fluorescence and EM images. Oligomers of C×43 form gap junctions on plasma membranes through which metabolites and signaling molecules are exchanged between cells. Tetracysteine tags have enabled pulse-chase experiments to observe the dynamic assembly and turnover of junctional plaque with minimal disturbance on C×43 structure and function. (A) Cellular C×43 is first „pulsed' with green-fluorescent FlAsH tag and then the nascent C×43 is „chased' with red-fluorescent ReAsH tag. (B) Nascent C×43 are observed to be added to periphery of the junctional plaque, indicated by the arrows. (C) Correlative EM shows high-resolution image of C×3 juncitional plaque (indicated by arrow) in the context of other subcellular structures. (B, C courtesy of R.Y. Tsien)

Figure 5

Cartoon and live cell imaging of the BirA-based peptide tag used to visualize internalization of low density lipoprotein (LDL) receptors. (A) LDL receptors are fused to biotin acceptor peptide (AP) and are expressed both inside and on the surface of the cell. Only cell surface receptors can be labeled with Alexa568 via monomerized streptavidin (mSA), which is not cell-permeable. After endocytosis, cell surface receptors are quenched with QSY quencher, such that internalized receptors can be selectively visualized. (B) Fluorescence images of cells immediately before and after surface fluorescence quenching (+QSY). With no incubation to allow for endocytosis, very few internalized receptors are detected. (C) With 5 min incubation at 37°C, some LDL receptors are internalized and thus protected from QSY quenching. (B, C courtesy of A.Y. Ting)