Introducing Bioorthogonal Functionalities into Proteins in Living Cells

Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted.

The green fluorescent protein (GFP) of Aquorea victoria is fluorescent when expressed as a recombinant protein in eukaryotic cells and has been used as a convenient marker of gene expression in vivo. It has also been used as a marker of the intracellular targeting of recombinant fusion proteins (part GFP, part protein of interest) which have been transiently expressed in eukaryotic cells grown in tissue culture. Thus, the use of GFP has proved a useful tool to study intracellular events in real-time. However, some transiently transfected cells fail to express, or localise correctly, the GFP-tagged protein. Therefore the production of stable cell lines expressing GFP-tagged integral membrane proteins may be essential for long-term studies. The generation of stably transfected eukaryotic cells expressing an integral membrane protein with a known, but poorly characterised intracellular trafficking pathway, would provide useful reagents for future, more precise, analysis of that pathway. TGN38 is a type I integral membrane protein which cycles between the trans-Golgi network (TGN) and cell surface; at steady state it is localised to the TGN. As such, TGN38 is an ideal candidate for tagging with GFP. We have generated cDNA constructs encoding ratTGN38 tagged at either the N- or C terminus with GFP. Transiently transfected rat (NRK) cells expressed active fluorophore, but failed to show correct localisation of the fusion protein. In contrast, both constructs are appropriately localised in stably transfected NRK cells and both are fluorescent. Furthermore, the recombinant GFP-tagged proteins and the endogenous TGN38 molecules show identical responses to drugs and temperature blocks known to perturb intracellular morphology and membrane traffic pathways. In fact morphological changes to the TGN induced by brefeldin A were observed at earlier time points than had been described previously using immunofluorescence analysis of fixed cells, thus validating the use of in vivo, real-time analysis of GFP-tagged proteins. In addition, we show that (in contrast to the situation in COS cells) elevated expression of ratTGN38 in NRK cells does not lead to a fragmentation of the TGN; this has implications for the role which TGN38 is playing in the maintenance of the morphology of the TGN. The data we present demonstrate that: (i) it is possible to generate stable cell lines expressing integral membrane proteins tagged with GFP; (ii) the GFP tag remains fluorescent when expressed on either the cytosolic or the lumenal side of all membranes of the secretory pathway up to and including that of the TGN; (iii) the GFP tag does not interfere with the transport of TGN38 along the secretory pathway or its retention in the TGN; (iv) GFP remains fluorescent in cells which have been processed for immunofluorescence analysis (using either paraformaldehyde or methanol fixation); and (v) TGN38 plays a role in maintaining the morphology of the TGN. Thus, stably transfected cells expressing GFP-tagged integral membrane proteins can be used as effective tools for the real-time study of intracellular morphology and membrane traffic pathways in eukaryotic cells.

RNA interference (RNAi) treatment of monkey COS-7 cells, a cell line that lacks nonmuscle myosin heavy chain II-A (NMHC II-A) but contains NMHC II-B and II-C, was used to investigate the participation of NMHC isoforms in cytokinesis. We specifically suppressed the expression of NMHC II-B or II-C using 21 nucleotide small interfering RNA (siRNA) duplexes. Down-regulation of NMHC II-B protein expression to 10.2 +/- 0.7% inhibited COS-7 cell proliferation by 50% in the RNAi-treated cells compared with control cells. Moreover, whereas 8.7 +/- 1.0% of control cells were multinucleated, 62.4 +/- 8.8% of the NMHC II-B RNAi-treated cells were multinucleated 72 h after transfection. The RNAi-treated cells had increased surface areas and, unlike control cells, lacked actin stress fibers. Treatment of the COS-7 cells with NMHC II-C siRNA decreased NMHC II-C expression to 5.2 +/- 0.1% compared with the endogenous content of II-C; however, down-regulation of NMHC II-C did not cause increased multinucleation. Immunoblot analysis using a pan-myosin antibody showed that the content of NMHC II-C was less than one-twentieth the amount of NMHC II-B, thereby explaining the lack of response to II-C siRNA. Introducing green fluorescent protein (GFP)-tagged NMHC II isoforms into II-B siRNA-treated cells resulted in reduction of multinucleation from 62.4 +/- 8.8% to 17.8 +/- 2.2% using GFP-NMHC II-B, to 29.8 +/- 7.4% using GFP-NMHC II-A, and to 34.1 +/- 8.6% using NMHC II-C-GFP. These studies have shown that expression of endogenous NMHC II-C in COS-7 cells is insufficient for normal cytokinesis and that exogenous NMHC II-A and NMHC II-C can, at least partially, rescue the defect in cytokinesis due to the loss of NMHC II-B.

Site-specific introduction of bioorthogonal handles into biomolecules provides powerful tools for studying and manipulating the structures and functions of proteins. Recent advances in bioorthogonal chemistry demonstrate that tetrazine-based bioorthogonal cycloaddition is a particularly useful methodology due to its high reactivity, biological selectivity, and turn-on property for fluorescence imaging. Despite its broad applications in protein labeling and imaging, utilization of tetrazine-based bioorthogonal cycloaddition has been limited to date by the requirement of a hydrophobic strained alkene reactive moiety. Circumventing this structural requirement, we report the site-specific incorporation of noncanonical amino acids (ncAAs) with a small isocyanide (or isonitrile) group into proteins in both bacterial and mammalian cells. We showed that under physiological conditions and in the absence of a catalyst these isocyanide-containing ncAAs could react selectively with tetrazine molecules via [4 + 1]-cycloaddition, thus providing a versatile bioorthogonal handle for site-specific protein labeling and protein decaging. Significantly, these bioorthogonal reactions between isocyanides and tetrazines also provide a unique mechanism for the activation of tetrazine-quenched fluorophores. The addition of these isocyanide-containing ncAAs to the list of 20 commonly used, naturally occurring amino acids expands our repertoire of reagents for bioorthogonal chemistry, therefore enabling new biological applications ranging from protein labeling and imaging studies to the chemical activation of proteins.

The utility of non-canonical amino acids in protein engineering has grown substantially over the past decade. Proteins containing these unnatural building blocks often have radically different biochemical or spectral characteristics than their wild-type counterparts. Furthermore, proteins may be endowed with chemical reactivity not found in the natural proteome upon the introduction of non-canonical amino acids. Successful incorporation of a non-canonical amino acid into recombinant proteins in E. coli is often dependent on engineering of the aminoacyl-tRNA synthetase (aaRS) activity of the cell. The bulk of the work described herein has focused on developing a system to rapidly screen libraries of mutant aaRS to identify clones capable of efficiently incorporating novel reactive non-canonical amino acids. The system is based on the display of reactive amino acid side chains on the surface of E. coli cells upon metabolic incorporation of the amino acid into recombinant outer membrane protein C (OmpC) and the subsequent covalent biotinylation of the reactive side chains. The cells are then stained with fluorescent avidin, thus rendering the cells incorporating the amino acid fluorescent and readily identifiable and sortable by flow cytometry. The feasibility of such a system was proven by incorporating the methionine surrogate azidohomoalanine (AHA) into OmpC and subsequently biotinylating the reactive azide groups via copper-catalyzed azide-alkyne ligation. Using an improved copper catalyst, low levels of incorporation of translationally inefficient amino acids azidoalanine, azidonorvaline, and azidonorleucine into OmpC were also detected. A saturation mutagenesis library of the methionyl-tRNA synthetase (MetRS) was designed, and cells transformed with this library were screened for the ability to incorporate the long chain amino acid azidonorleucine into recombinant proteins efficiently. Several MetRS mutants were identified with such activity using the cell surface display system. MetRS containing a single amino acid mutation, leucine 13 to glycine (L13G) that occurs in each of the three mutants discovered in the screen, is very efficient at incorporating azidonorleucine into proteins. In the last part of the work described in this thesis, azidohomoalanine was used to tag newly-synthesized proteins in mammalian cells, thus endowing the newly-synthesized proteins with unique bioorthogonal chemical reactivity. Following covalent biotinylation via the azide-alkyne ligation, these proteins could be selectively enriched for by avidin chromatography and identified using shotgun proteomic approaches. Nearly 200 newly-synthesized proteins were identified unequivocally in just a two-hour window. This technique promises to develop into a highly useful tool for the examination of proteome dynamics.

Direct delivery of proteins into mammalian cells is a challenging problem in biological and biomedical applications. The most common strategies for the delivery of proteins into the cells include the use of cell-penetrating peptides or supercharged proteins. Herein, we show for the first time that a single atom change, hydrogen to halogen, at one of the tyrosine residues can increase the cellular entry of ∼28 kDa green fluorescent protein (GFP) in mammalian cells. The protein uptake is facilitated by a receptor-mediated endocytosis and the cargo can be released effectively into cytosol by co-treatment with the endosomolytic peptide ppTG21.

The bioorthogonal chemistry has become a valuable tool for specific labeling of biomolecules in living systems. One emerging example is to convert the palladium-mediated carbon-carbon bond formation reaction, a landmark reaction in organic synthesis, into the biocompatible reaction for biomolecule modifications. A prerequisite to enable such reactions to be executed on biological samples is to incorporate the corresponding bioorthogonal functional groups into target biomolecules. The genetic code expansion strategy is a leading technology on introducing diverse bioorthogonal functionalities into pro- teins, and the most recently developed pyrrolysine system can now allow unnatural amino acids (UAAs) bearing bioorthogo- nal handles into proteins in prokaryotic cells, eukaryotic cells and even in living animals. In this perspective, we will first introduce some of the forefronts of the bioorthognal reactions as well as the genetic code expansion strategy. We then will discuss the advantages and attractiveness of combining these newly emerged tools, Pd-mediated biomolecular labeling and pyrrolysine-based genetic code expansion system in particular, to specifically label and study proteins in living mammalian cells. More biocompatible reactions and more flexible UAA incorporation system will significantly strengthen and expand our ability to specifically manipulate proteins in their native cellular environment. Keywords protein labeling; bioothorgonal; palladium catalyzed; cross coupling; unnatural amino acid

BackgroundThe tagging strategy enables full-length endogenous proteins in mammalian cells to be expressed as green fluorescent fusion proteins from their authentic promoters.ResultsWe describe improved genetic tools to facilitate protein tagging in mammalian cells based on a mobile genetic element that harbors an artificial exon encoding a protein tag. Insertion of the artificial exon within introns of cellular genes results in expression of hybrid proteins consisting of the tag sequence fused in-frame to sequences of a cellular protein. We have used lentiviral vectors to stably introduce enhanced green fluorescent protein (EGFP) tags into expressed genes in target cells. The data obtained indicate that this strategy leads to bona fide tripartite fusion proteins and that the EGFP tag did not affect the subcellular localization of such proteins.ConclusionThe tools presented here have the potential for protein discovery, and subsequent investigation of their subcellular distribution and role(s) under defined physiological conditions, as well as for protein purification and protein-protein interaction studies.

Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein

Site‐specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl‐tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA‐suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual‐ncAA incorporation. Using these platforms, we site‐specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site‐specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

Analysis of the developing proteome has been complicated by a lack of tools that can be easily employed to label and identify newly synthesized proteins within complex biological mixtures. Here, we demonstrate that the methionine analogs azidohomoalanine and homopropargylglycine can be globally incorporated into the proteome of mice through facile intraperitoneal injections. These analogs contain bio-orthogonal chemical handles to which fluorescent tags can be conjugated to identify newly synthesized proteins. We show these non-canonical amino acids are incorporated into various tissues in juvenile mice and in a concentration dependent manner. Furthermore, administration of these methionine analogs to pregnant dams during a critical stage of murine development, E10.5–12.5 when many tissues are assembling, does not overtly disrupt development as assessed by proteomic analysis and normal parturition and growth of pups. This successful demonstration that non-canonical amino acids can be directly administered in vivo will enable future studies that seek to characterize the murine proteome during growth, disease and repair.

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

The efficient, site-specific introductionof unnatural amino acidsinto proteins in mammalian cells is an outstanding challenge in realizingthe potential of genetic code expansion approaches. Addressing thischallenge will allow the synthesis of modified recombinant proteinsand augment emerging strategies that introduce new chemical functionalitiesinto proteins to control and image their function with high spatialand temporal precision in cells. The efficiency of unnatural aminoacid incorporation in response to the amber stop codon (UAG) in mammaliancells is commonly considered to be low. Here we demonstrate that tRNAlevels can be limiting for unnatural amino acid incorporation efficiency,and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammaliancells. In addition, we engineer eRF1, that normally terminates translationon all three stop codons, to provide a substantial increase in unnaturalamino acid incorporation in response to the UAG codon without increasingreadthrough of other stop codons. By combining the optimized pyrrolysyl-tRNAsynthetase/tRNACUA expression system and an engineeredeRF1, we increase the yield of protein bearing unnatural amino acidsat a single site 17- to 20-fold. Using the optimized system, we produceproteins containing unnatural amino acids with comparable yields toa protein produced from a gene that does not contain a UAG stop codon.Moreover, the optimized system increases the yield of protein, incorporatingan unnatural amino acid at three sites, from unmeasurably low levelsup to 43% of a no amber stop control. Our approach may enable theefficient production of site-specifically modified therapeutic proteins,and the quantitative replacement of targeted cellular proteins withversions bearing unnatural amino acids that allow imaging or syntheticregulation of protein function.

For use in site-specific bioorthogonal labeling of expressed G protein-coupled receptors (GPCRs) in live cells, we developed a luciferase-based reporter assay. The assay was used to compare amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used the assay system to compare side-by-side the efficiency of incorporation of three different noncanonical amino acids [4-azido-l-phenylalanine (azF), cyclopropene-l-lysine (CpK), and trans-cyclooct-2-en-l-lysine (TCOK)] at three different sites on a GPCR using three different genetic code expansion plasmid systems. As a model GPCR, we engineered an epitope-tagged C-C chemokine receptor 5 (CCR5)-RLuc3 fusion for expression in HEK293T cells. Satisfactory incorporation of azF, CpK, and TCOK into heterologously expressed CCR5 was achieved. We also carried out cell-based calcium mobilization assays to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered mutants using heterobivalent compounds containing bioorthogonal tethering groups linked to either a small-molecule fluorophore or a peptide. Favorable reaction kinetics of tetrazine-containing compounds with CCR5 harboring TCOK was observed. However, bioorthogonal labeling in live cells of CCR5 harboring CpK with tetrazine-containing compounds using the inverse electron demand Diels-Alder ligation was overall slightly more efficient than other reactions tested.

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.