MiniVIPER Is a Peptide Tag for Imaging and Translocating Proteins in Cells

Abstract

Microscopy allows researchers to interrogate proteins within a cellular context. To deliver protein-specific contrast, we developed a new class of genetically encoded peptide tags called versatile interacting peptide (VIP) tags. VIP tags deliver a reporter to a target protein via the formation of a heterodimer between the peptide tag and an exogenously added probe peptide. We report herein a new VIP tag named MiniVIPER, which is comprised of a MiniE–MiniR heterodimer. We first demonstrated the selectivity of MiniVIPER by labeling three cellular targets: transferrin receptor 1 (TfR1), histone protein H2B, and the mitochondrial protein TOMM20. We showed that either MiniE or MiniR could serve as the genetically encoded tag. Next, we demonstrated MiniVIPER’s versatility by generating five spectrally distinct probe peptides to label tagged TfR1 on live cells. Lastly, we demonstrated two new applications for VIP tags. First, we used MiniVIPER in combination with another VIP tag, VIPER, to selectively label two different proteins in a single cell (e.g., TfR1 with H2B or TOMM20). Second, we used MiniVIPER to translocate a fluorescent protein to the nucleus through in situ dimerization of mCherry with H2B-mEmerald. In summary, MiniVIPER is a new peptide tag that enables multitarget imaging and artificial dimerization of proteins in cells.

Graphical Abstract

Fluorescence microscopy (FM) is a valuable resource for studying protein functions and interactions within a native cellular environment. Multicolor FM observations of proteins or subcellular structures are enabled by small molecule stains, immunolabeling, and genetically encoded tags.^1–3 Among genetically encoded tags, fluorescent protein tags [e.g., green fluorescent protein (GFP)] are ubiquitous in FM imaging. However, fluorescent proteins have disadvantages, including their large (~30 kDa) size. Some variants oligomerize at high concentrations, which can alter the function of the protein under study.⁴ Additionally, the fluorescent protein chromophore has limitations on its spectral properties, including the brightness, photostability, and Stokes shift. This last issue was addressed by newer protein tags, such as the SNAP-Tag⁵ and HaloTag,⁶ which form a covalent bond with a fluorescent ligand that can be synthesized with optimal fluorescence properties.⁷ However, these tags are still large: SNAP-Tag is 22 kDa,⁵ and HaloTag is 33 kDa.⁶

An ideal genetically encoded tag should be small while retaining labeling specificity. The smallest peptide tags are the tetracysteine tag^8,9 and tetraserine tag,¹⁰ which bind to fluorogenic reporters. A few other approaches modify a peptide tag sequence via an enzyme modification. In this category, tags are modified post-translationally by biotin ligase,¹¹ lipoic acid ligase,¹² phosphopantetheinyl transferases,¹³ or other enzymes.¹⁴

A separate class of genetically encoded peptide tag labels target proteins using heterodimeric coiled coils.¹⁵ For these tags, the protein of interest is fused to a short (3–6 kDa) peptide that forms a tight heterodimer with a fluorophore-conjugated peptide.¹⁵ This approach was first used in 2008 by Yano and co-workers to image membrane receptors using an E3–K3 heterodimer.¹⁶ Coiled-coil peptides have since been used to deliver fluorogenic probes,^17–19 mediate proximity-induced reactions,^19–23 localize proteins,²⁴ or regulate transcription. ²⁴

Our contribution to this area was the development of versatile interacting peptide (VIP) tags. VIP tags enable specific protein labeling in live and fixed cells. VIP Y/Z, consisting of a CoilY–CoilZ dimer, was the first VIP tag published for selectively labeling protein targets in cell lysates and on living cells.²⁵ We then developed a tag named VIPER, comprised of a heterodimer between a CoilE tag (5.2 kDa) and a CoilR probe peptide. We demonstrated that VIPER is a useful tag for correlative light and electron microscopy and for quantifying cell receptors in micrographs.²⁶

In the current work, we designed a modified version of VIPER that retains protein labeling specificity in a smaller size. We named this tag MiniVIPER, and it is comprised of a heterodimeric coiled coil between two peptides: MiniE and MiniR. Both peptides are small (4.3 kDa), and either one can serve as the genetic tag. We showed that MiniVIPER enables the selective protein labeling of cellular targets. We illustrated the versatility of MiniVIPER by imaging a cell receptor using five spectrally distinct fluorophores spanning green to far-red emission. We then demonstrated that this tag enables two useful applications. First, we expressed two VIP-tagged proteins within a single cell and demonstrated the selective labeling of both protein targets. Second, we used MiniVIPER to translocate a protein to the nucleus.

MATERIALS AND EXPERIMENTAL DETAILS

See the Supporting Information for a detailed description of all materials and methods used.

RESULTS AND DISCUSSION

Design of MiniVIPER.

VIP tags are comprised of α-helical coiled coils, with each coil consisting of heptad repeats denoted abcdefg. The a and d residues form a hydrophobic core between the two coils, while charged residues at positions e and g form salt bridges (g↔e′). Both VIPER and MiniVIPER have a series of E↔R salt bridges, which form stronger interhelical interactions than the commonly used E↔K bridges²⁷ (Figure 1).

Figure 1.

Sequence comparison of VIPER and MiniVIPER. Helical wheel diagrams of (A) VIPER and (B) MiniVIPER were generated using DrawCoil 1.0 (https://grigoryanlab.org/drawcoil/). Charged residues are colored red or blue, with salt bridges indicated by dashed lines. Polar residues are colored orange, and hydrophobic residues are colored gray. Residues altered in MiniVIPER are denoted with an asterisk. (C) Sequences of VIP tags and probes, with coil-forming amino acids in bold. The fifth heptad in CoilE and CoilR, which was removed in MiniE and MiniR, is indicated by a dashed line. Other changes to the MiniVIPER sequences are underlined in green.

We designed MiniVIPER by removing the last (fifth) heptad of the previously published VIPER tag.²⁶ We considered the effect this change would have on dimer stability. Generally, removing a heptad reduces both the melting temperature (T_m) and the dissociation constant (K_D) in coiled-coil dimers.^28–30 For example, De Crescenzo and co-workers systematically assessed the effect of chain length on coiled-coil binding.²⁸ They found that going from five to four heptads in a model heterodimer resulted in a 100-fold loss of affinity, from 6.3 × 10⁻¹¹ to 7.3 × 10⁻⁹ M. Further reduction, to three heptads, made the pair semi-unstable (K_D = 3.2 × 10⁻⁵ M). Thomas and co-workers examined a different heterodimer and observed a >30000-fold loss of affinity going from four to three heptads.³⁰ Although three heptad coiled coils have been used successfully to label proteins (e.g., the E3–K3 dimer¹⁶), we opted to retain four heptads in MiniVIPER. Compared to VIPER (K_D = 1.3 × 10⁻¹¹ M; T_m = 73 °C), we predict that MiniVIPER exhibits an ~100-fold reduction in K_D, which we considered an acceptable trade-off for a decrease in size.

In addition to reducing the length, we made a few other modifications at once to create MiniVIPER (Figure 1B). First, we improved the charge balance by making a g-position mutation in MiniE (Arg32Glu) and MiniR (Glu32Arg); these residues do not form a salt bridge. For the MiniE tag, this decreased the isoelectric point (pI) dramatically compared to that of the CoilE tag. All other charged residues were retained at the e and g positions to discourage the formation of homodimers. An alanine in the fifth position on MiniR was changed to a valine (Ala5Val) to strengthen binding.³¹ Overall, MiniVIPER has eight E↔R salt bridges, an Asn-Asn match in the second heptad, and a hydrophobic interface, all features that promote parallel heterodimer formation.^32–34

Production of the MiniE and MiniR Probe Peptides.

Probe peptides have a C-terminal histidine tag, (His)₆, for purification by immobilized metal affinity chromatography (IMAC).³⁵ The probe peptides additionally include a flexible linker, (Gly)₃-(Ala)₃,³⁶ followed by the sequence Trp-Gly-Leu-Cys-Tyr-Pro-Trp-Val-Tyr-Gly, which enhanced the properties of the probe peptides in three ways. First, the addition of aromatic residues increased the extinction coefficient by >10-fold, improving the accuracy of quantification by absorbance measurements. Second, inclusion of a dibenzocyclooctyne (DBCO) tag (Leu-Cys-Tyr-Pro-Trp-Val-Tyr) enabled site-specific thiol-maleimide or thiol-yne³⁷ conjugation reactions at the Cys residue. Third, we found that adding this sequence increased the yield of the recombinant peptide expressed in BL21-DE3 cells.

It is feasible to make or purchase fluorophore-modified peptides via solid-phase peptide synthesis. However, we instead used standard methods to express and purify the MiniE probe and MiniR probe, as described in the Supporting Information. We modified MiniE with a TAMRA-cyclooctyne via thiol-yne conjugation and obtained MiniE-TAMRA (16% labeled). The remaining probe peptides were made via thiol-maleimide chemistry:³⁸ MiniE-Cy5 (59%), MiniE-biotin (100%), MiniE-Cy3 (52%), MiniE-OregonGreen (OG) 488 (64%), MiniR-Cy5 (29%), and MiniR-biotin (100%). CoilR and CoilY peptides were made as previously described: CoilR-Cy5 (90%),²⁶ CoilR-AF488 (45%),³⁵ CoilY-Cy5 (50%),²⁵ and CoilY-biotin.²⁵

Observation of MiniVIPER-Labeled Proteins in Cells.

In initial experiments, we confirmed that MiniVIPER enabled selective protein labeling in cells (Figure 2). First, we imaged transferrin receptor 1 (TfR1) by FM. We selected TfR1 because it has well-characterized membrane localization, transferrin binding, and rapid internalization via clathrin-mediated endocytosis.^39,40 We transfected Chinese hamster ovary (CHO) TRVb (ΔTfR1 ΔTfR2) cells⁴¹ to express untagged (TfR1) or tagged (TfR1-miniR or TfR1-miniE) receptors. Live cells were cooled to halt endocytosis and then treated with the probe peptide and fluorescent transferrin-AF488 (Tf-AF488) to counterstain the receptor. Cells were fixed before being imaged by confocal FM (Figure 2B). Micrographs showed selective labeling of TfR1-miniR with MiniE-Cy5. We observed a Cy5 signal at the cell surface that co-localized with the Tf-AF488 signal. The absence of MiniE-Cy5 labeling in cells expressing untagged TfR1 confirmed the specificity of labeling. We found that either MiniR or MiniE could serve as the genetically encoded tag. TfR1-miniE was labeled with MiniR-Cy5, and receptor–ligand co-localization was observed by confocal FM. Although micrographs were taken postfixation, prior studies showed that VIP tags are compatible with live-cell imaging.^25,26,42

Figure 2.

MiniVIPER enabled selective fluorophore labeling of cellular proteins. (A) Representation of the tagged constructs used to label: a cell receptor (TfR1), the nucleus (H2B), and the mitochondria (TOMM20). (B) CHO TRVb cells expressing TfR1, TfR1-miniR, or TfR1-miniE were treated live on ice with Tf-AF488 and either MiniE-Cy5 (top) or MiniR-Cy5 (bottom). Cells were fixed before being imaged. U-2 OS cells expressing (C) H2B-mEmerald, H2B-MiniR-mEmerald, or H2B-MiniE-mEmerald or (D) TOMM20-mCherry, TOMM20-miniR-mCherry, or TOMM20-miniE-mCherry were treated postfixation with MiniE-Cy5 (top) or MiniR-Cy5 (bottom). AF488, mEmerald, and mCherry signals are falsely colored green. The Cy5 signal is falsely colored magenta. The nuclear stain (blue) is shown in the channel merge, and magenta-green overlap appears white. Protein sequences are listed in Table S1.

Next, we imaged two subcellular structures: the nucleus (Figure 2C) and mitochondria (Figure 2D). We imaged tagged and untagged histone 2B-mEmerald (H2B-mEmerald) and translocase of outer mitochondrial membrane 20-mCherry (TOMM20-mCherry) in transfected human osteosarcoma (U-2 OS) cells. Cells were fixed and permeabilized before being treated with Cy5-conjugated probe peptides. MiniVIPER labeling was specific, and Cy5 fluorescence was observed only in cells expressing tagged targets. For VIP-tagged proteins, we observed co-localization of the Cy5 signal with the fluorescent protein (e.g., mCherry with Cy5 in the mitochondria or mEmerald with Cy5 in the nucleus). Protein localization and organelle morphology appeared to be unaltered between MiniVIPER-tagged and untagged proteins.

Assessment of VIPER and MiniVIPER Cross-Reactivity.

VIPER and MiniVIPER share four of five heptad sequences (see Figure 1), and we anticipated that we might detect cross-reactivity between them. We collected fluorescent micrographs to evaluate pairwise combinations of the peptide tags with Cy5-labeled probe peptides, as shown schematically in Figure 3A. As expected from their sequences, we observed labeling of the CoilE tag with both CoilR-Cy5 and MiniR-Cy5 (Figure 3B–D). This is consistent with the formation of the predicted coiled-coil pairs with a hydrophobic interface and optimal E↔R salt bridges. Similarly, the MiniE tag was labeled by both “R” probe peptides. Homodimers (e.g., MiniR–MiniR or MiniE–MiniE) were not observed, including for the CoilE tag with MiniE-Cy5. In other words, target protein labeling was observed for only E–R pairs, not for E–E or R–R pairs. On the basis of these results, we conclude that both CoilE and MiniE dimerize with both CoilR and MiniR.

Figure 3.

MiniVIPER and VIPER cross-react predictably to form heterodimers. (A) Schematic of the predicted charge interactions between various tag and probe peptide (coiled-coil) combinations. (B–D) Cells were transfected to express untagged or tagged (B) TfR1, (C) H2B-mEmerald, or (D) TOMM20-mCherry. The peptide tag is indicated to the left of the micrographs. Cells were treated live (B) or postfixation (C and D) with CoilR-Cy5, MiniR-Cy5, or MiniE-Cy5, as indicated at the top of each column. Micrographs for each target protein were acquired and processed with identical settings to enable direct comparison between pairs. For a few pairs, the Cy5 signal was above the background but had to be autoscaled to be observed (see the insets). The complete data sets can be found in Figures S1–S3.

MiniVIPER: One Tag, Five Fluorophores.

VIP tags confer a practical advantage for imaging applications in that the peptide tag can be detected with various spectrally distinct reporter peptides. We demonstrated this feature in cells expressing TfR1-miniR and labeled with one of five MiniE probe peptides (Figure 4). Micrographs showed that TfR1-miniR could be labeled live with spectrally distinct fluorophores with green to far-red emission. Among these, Cy5 is a preferred fluorophore for super-resolution microscopy.⁴³ Qdot655 is bright, fluorescent, and photostable, making it ideal for single-particle tracking or detecting low-abundance targets. Additionally, Qdots have an electron dense core for imaging by correlative light and electron microscopy.^44,45 Peptides were attached to commercial fluorophores using either thiol-maleimide chemistry or thiol-yne chemistry, as described above. Other colors should be readily accessible for multicolor imaging applications. Moreover, probe peptides made by solid-phase peptide synthesis could contain novel features, such as bioorthogonal conjugation sites (e.g., an alkyne handle), a modified backbone, linkers, or fluorescent amino acids.

Figure 4.

VIP-tagged protein can be labeled with a variety of fluorophores. Live CHO TRVb cells expressing TfR1-miniR were treated with fluorescent transferrin and one of five probe peptides, as indicated. Cells were fixed before being imaged. (A) MiniE-OG488 [excitation (ex) at 501 nm, emission (em) at 526 nm, quantum yield (QY) of 0.92]. (B) MiniE-Cy3 (ex at 548 nm, em at 563 nm, QY of 0.14). (C) MiniE-TAMRA (ex at 552 nm, em at 571 nm, QY of 0.41). (D) MiniE-Cy5 (ex at 646 nm, em at 662 nm, QY of 0.18). (E) MiniE-biotin and streptavidin-Qdot655 (ex at 405 nm, em at 655 nm, QY of 1.0). (F) Predicted emission spectra for the indicated fluorophores were generated using SpectraViewer (www.thermofisher.com).

Combining VIP Tags to Image Two Distinct Cellular Targets.

Many new discoveries in cell biology have been enabled by imaging proteins using genetically encoded tags. Most of these studies rely on spectrally distinct probes to label more than one target,¹ which enables proteins to be examined relative to each other and in relationship to their environment. In prior work, we demonstrated that VIP tags could be imaged alongside other targets labeled with small molecule stains (e.g., Hoechst), fluorescent ligands (e.g., transferrin), fluorescent proteins, or antibodies.^25,26

Now we show that VIP tags can be combined to label two distinct protein targets in one cell. We opted to label proteins localized to distinct subcellular regions to ensure that the two VIP-tagged proteins would not dimerize with each other. First, we used MiniVIPER with VIPER to label two targets: TfR1-miniR with H2B-CoilE. In co-transfected CHO TRVb cells, we labeled TfR1-miniR live (with MiniE-Cy5), fixed cells and then treated them with CoilR-AF488 to label H2B-CoilE in the nucleus. In cells co-expressing both targets, we observed selective MiniVIPER labeling at the cell surface and VIPER labeling in the nucleus, as anticipated (Figure 5A). We considered the possibility that TfR1-miniR could heterodimerize with H2B-CoilE but found no evidence of TfR1 in the nucleus by MiniVIPER labeling or by immunolabeling (Figure S4). These experiments demonstrate that the MiniR tag and the CoilE tag can be used together to detect two targets in one cell.

Figure 5.

VIP tags can be combined to image two targets simultaneously. (A) CHO TRVb cells were treated with MiniE-Cy5 (top), CoilR-AF488 (middle), or both MiniE-Cy5 and CoilR-AF488 (bottom). (B) U-2 OS cells were treated with MiniE-Cy5 (top), CoilR-AF488 (middle), or both MiniE-Cy5 and CoilR-AF488 (bottom). Cells were fixed before being imaged, and micrographs were falsely colored (Cy5, magenta; AF488, green; mCherry, red; Hoechst 33342, blue).

Next, we imaged TfR1-miniR with TOMM20-CoilE-mCherry in U-2 OS cells. Co-transfected cells were labeled live with MiniE-Cy5, fixed, and then treated with CoilR-AF488. In cells expressing both tagged proteins, we observed VIPER labeling (CoilR-AF488 signal) localized to the mitochondria and co-localized with mCherry (Figure 5B). In the same cells, MiniVIPER labeling (MiniE-Cy5 signal) was observed at the cell surface and in endosomes, consistent with TfR1 labeling. These results further establish that MiniVIPER and VIPER can be used together to label two distinct targets.

Lastly, we imaged cells expressing proteins labeled with VIP Y/Z, a previously published VIP tag,²⁵ with proteins labeled with MiniVIPER or VIPER. Again, we observed selective labeling of each protein target (Figure S5).

One caveat of these studies is that some tag combinations could induce an artificial interaction if the two VIP-tagged target proteins (with interacting peptide coils) come into the proximity of each other. We selected our targets (e.g., nucleus with the cell membrane or mitochondria with the cell membrane) to circumvent this potential issue. On the basis of their sequences, we do not expect to observe cross-binding of VIP Y/Z with either VIPER or MiniVIPER.

VIP-Mediated Translocation of Proteins.

The activities of many cellular proteins are controlled by localization, protein–protein interactions, and the movement of a protein between subcellular locations (i.e., translocation). Chemically induced dimerization (CID) systems are often used to study these processes inside cells.^46–48 Another method, bimolecular fluorescence complementation, uses split fluorescent proteins to detect protein–protein interactions.⁴⁹ A third approach detects a translocated protein through binding (e.g., via a coiled-coil dimer) between the target protein and a fluorescent protein.^24,50

Compared to other systems, the benefits of using VIP tags for protein translocation include their small size, directable orientation, stability, and independence of exogenous reagents. With that in mind, we sought to demonstrate that MiniVIPER could be used to translocate proteins in living cells. First, cells were transfected to express soluble MiniR-mCherry in the cytosol with an untagged H2B-mEmerald. We observed red fluorescence throughout the interior of cells, while green fluorescence was observed in only the nuclei (Figure 6A, top row). Next, cells were transfected to express both MiniR-mCherry and H2B-MiniE-mEmerald. We observed a remarkable difference. Upon expression of the MiniE-tagged protein, red fluorescence was observed solely in the nucleus and co-localized with the green fluorescent signal (Figure 6A, bottom row). This indicated to us that the formation of the MiniR–MiniE dimer resulted in the localization of mCherry to the nucleus. Analogous experiments confirmed that the opposite orientation (e.g., using MiniE-mCherry with H2B-MiniR-mEmerald) or VIPER also enabled VIP-mediated protein translocation in living cells (Figure 6B,C).

Figure 6.

Translocation of mCherry to the nucleus using VIP-mediated protein dimerization. (A) Cells expressing H2B-mEmerald and MiniR-mCherry (top row) or H2B-MiniE-mEmerald and MiniR-mCherry (bottom row) imaged after fixation. Under the latter condition, formation of the MiniVIPER dimer translocated mCherry to the nucleus, with green-magenta co-localization apparent in white. (B) Cells expressing H2B-mEmerald and MiniE-mCherry (top row) or H2B-MiniR-mEmerald and MiniE-mCherry (bottom row). (C) VIPER-mediated translocation of CoilR-mCherry to the nucleus in the presence of H2B-CoilE-mEmerald. Analogous experiments were conducted and imaged in live cells (Figure S6). (D) FRET efficiency for cells expressing tagged mCherry with untagged (empty symbols) or coil-tagged (filled symbols) H2B-mEmerald (mEm). The FRET efficiency was measured in 10 nuclei per condition (Table S7), and error bars represent the standard error of the mean (***p < 0.001; ****p < 0.0001).

Next, we quantified these VIP-mediated interactions using Förster resonance energy transfer (FRET) imaging. Green and red fluorescent proteins are established donor–acceptor FRET pairs, although the FRET efficiency is typically low (4–29%).^51,52 In our system, described above, we anticipated that mEmerald and mCherry would be close enough (<10 nm) after VIP-mediated dimerization to be detectable by FRET. We opted to use acceptor photobleaching (AP)-FRET, which measures the increase in donor fluorescence after acceptor photobleaching. The advantage of using AP-FRET is that the FRET efficiency can be determined directly.⁵³ We observed FRET efficiencies ranging from 9% to 13% for cells with VIP-induced mEmerald-mCherry dimers in the nucleus (Figure 6D). By comparison, when H2B-mEmerald (no VIP tag) was co-expressed in cells with tagged mCherry, the FRET efficiency ranged from −0.9 ± 0.5% to 3.0 ± 1.5%. Negative FRET can be attributed to noise from the detectors and background fluorescence from the sample.⁵⁴

These studies show that VIP tags can be used to dimerize two proteins together and that this interaction can be measured using AP-FRET. Overall, these studies demonstrate that VIP tags are effective tools for translocating proteins inside cells.

CONCLUSION

In this work, we introduced MiniVIPER as a small and specific genetically encoded peptide tag compatible with multiple imaging applications. MiniVIPER enabled fluorophore labeling of receptors (TfR1) on live cells or histones (H2B-mEmerald) and mitochondria (TOMM20-mCherry) in fixed cells. Labeling was target-specific, and VIP tagging did not interfere with protein localization. At just 4.3 kDa, both the MiniE and the MiniR tags are far smaller than fluorescent proteins and most other tags, even after formation of the coiled-coil dimer. Another distinguishing feature is the fact that VIP tags can be detected with a variety of spectrally distinct probe peptides without changing the genetic tag.

We demonstrated two new applications for VIP tags. First, we established the generality of using a combination of two VIP tags for multicolor imaging. We used FM to observe MiniVIPER-labeled TfR1 with either VIPER-labeled H2B or VIPER-labeled TOMM20-mCherry. Furthermore, we demonstrated that the VIP Y/Z tag can be observed together with either VIPER or MiniVIPER. We have not yet combined all three VIP tags together, but that is a logical next step.

In a second application, we used VIP tags to alter the subcellular localization of a target protein. Specifically, we used MiniVIPER to translocate cytosolically expressed mCherry to the nucleus. Interactions were quantified using AP-FRET, which confirmed that VIP-mediated dimerization brought the two target proteins into the proximity of each other.

The versatility of VIP tags makes them amenable to different applications with thoughtful experimental design. VIP tags are unique in their ability to enable selective protein labeling, multitarget imaging, and translocation. We believe that these features make MiniVIPER a beneficial and useful addition to the cell biologist’s toolbox.