Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors

Chemical modification of proteins has a rich history in biochemistry and chemical biology. However, studies of membrane protein function, especially in cases where functional expression is low and purification and reconstitution are not feasible, present unique challenges. Heptahelical G-protein-coupled receptors (GPCRs) are a particularly important class of cell-surface receptors that represent targets of more than a quarter of all therapeutic drugs. Understanding with chemical precision how GPCRs function in biological membranes remains a central problem in biology. Recently a number of creative strategies have been developed that allow site-specific attachment of chemical probes or tags directly on expressed receptors or on biologically active peptide ligands or substrates. One particularly important advance is the genetic encoding of unnatural amino acids (UAAs) with unique small bioorthogonal tags using amber codon suppression in mammalian cells. This method should allow site-specific labeling of GPCRs with various molecular probes to facilitate cell-based studies of protein-protein or protein-ligand interactions and the visualization of conformational changes using fluorescence spectroscopy or single-molecule imaging.     

Genetically encoded protein labeling and crosslinking in living Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is a major human pathogen for hospital-acquired infections. We report the genetic code expansion of this opportunistic pathogen by using the pyrrolysyl-tRNA synthetase-tRNA system, which enabled the genetic and site-specific incorporation of unnatural amino acids bearing bioorthogonal handles or photo-affinity groups into proteins in PA. This strategy allowed us to conduct bioorthogonal labeling and imaging of flagella, as well as site-specific photo-affinity capturing of interactions between a Type III secretion effector and its chaperone inside living bacteria.     

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.     

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

     

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.     

Utilization of alkyne bioconjugations to modulate protein function

The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.     

Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.     

Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells

The genetic code of living organisms has been expanded to allow the site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon UAG. Numerous amino acids have been incorporated including photo-crosslinkers, chemical handles, heavy atoms and post-translational modifications, and this has created new methods for studying biology and developing protein therapeutics and other biotechnological applications. Here we describe a protocol for reprogramming the amino-acid substrate specificity of aminoacyl-tRNA synthetase enzymes that are orthogonal in eukaryotic cells. The resulting aminoacyl-tRNA synthetases aminoacylate an amber suppressor tRNA with a desired unnatural amino acid, but no natural amino acids, in eukaryotic cells. To achieve this change of enzyme specificity, a library of orthogonal aminoacyl-tRNA synthetase is generated and genetic selections are performed on the library in Saccharomyces cerevisiae. The entire protocol, including characterization of the evolved aminoacyl-tRNA synthetase in S. cerevisiae, can be completed in approximately 1 month.     

无细胞体系非天然蛋白质合成研究进展

无细胞非天然蛋白质合成作为蛋白质研究的新兴手段,已成功用于表征蛋白质分子间、蛋白质与核酸分子间相互作用等基础科学研究及医药蛋白、蛋白质材料等工业生产领域。无细胞非天然蛋白质合成系统不需维持细胞的生长,无细胞膜阻碍,可依据研究目的添加基因元件或化学物质从而增强工程设计和过程调控的自由性;也可赋予蛋白质新的特性、结构及功能,如可实现蛋白翻译后修饰、反应手柄引入、生物物理探针及多聚蛋白质合成等。文中系统地综述了目前应用于无细胞蛋白质合成系统中的非天然氨基酸嵌入方法,包括全局抑制及基于正交翻译体系的终止密码子抑制、移码抑制、有义密码子再分配和非天然碱基等方法的研究进展,及非天然氨基酸在蛋白质修饰、生物物理探针、酶工程、蛋白质材料以及医药蛋白质生产等领域的应用进展,并分析了该体系的发展前景及广泛工业化应用的机遇与挑战。     

Synthesis of Non-linear Protein Dimers through a Genetically Encoded Thiol-ene Reaction

Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylT_CUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins.     

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

We developed a general approach that allows unnatural amino acids with diverse physicochemical and biological properties to be genetically encoded in mammalian cells. A mutant Escherichia coli aminoacyl-tRNA synthetase (aaRS) is first evolved in yeast to selectively aminoacylate its tRNA with the unnatural amino acid of interest. This mutant aaRS together with an amber suppressor tRNA from Bacillus stearothermophilus is then used to site-specifically incorporate the unnatural amino acid into a protein in mammalian cells in response to an amber nonsense codon. We independently incorporated six unnatural amino acids into GFP expressed in CHO cells with efficiencies up to 1 mug protein per 2 x 10(7) cells; mass spectrometry confirmed a high translational fidelity for the unnatural amino acid. This methodology should facilitate the introduction of biological probes into proteins for cellular studies and may ultimately facilitate the synthesis of therapeutic proteins containing unnatural amino acids in mammalian cells.     

Chemical mutagenesis: selective post-expression interconversion of protein amino acid residues

The ability to alter protein structure by site-directed mutagenesis has revolutionized biochemical research. Controlled mutations at the DNA level, before protein translation, are now routine. These techniques allow specific, high fidelity interconversion largely between 20 natural, proteinogenic amino acids. Nonetheless, there is a need to incorporate other amino acids, both natural and unnatural, that are not accessible using standard site-directed mutagenesis and expression systems. Post-translational chemistry offers access to these side chains. Nearly half a century ago, the idea of a 'chemical mutation' was proposed and the interconversion between amino acid side chains was demonstrated on select proteins. In these isolated examples, a powerful proof-of-concept was demonstrated. Here, we revive the idea of chemical mutagenesis and discuss the prospect of its general application in protein science. In particular, we consider amino acids that are chemical precursors to a functional set of other side chains. Among these, dehydroalanine has much potential. There are multiple methods available for dehydroalanine incorporation into proteins and this residue is an acceptor for a variety of nucleophiles. When used in conjunction with standard genetic techniques, chemical mutagenesis may allow access to natural, modified, and unnatural amino residues on translated, folded proteins.     

Synthetase polyspecificity as a tool to modulate protein function

The site-specific incorporation of unnatural amino acids (UAAs) into proteins in bacteria is made possible by the evolution of aminoacyl-tRNA synthetases that selectively recognize and aminoacylate the amino acid of interest. Recently we have discovered that some of the previously evolved aaRSs display a degree of polyspecificity and are capable of recognizing multiple UAAs. Herein we report the polyspecificity of an aaRS evolved to encode a comarin containing amino acid. This polyspecificity was then exploited to introduce several UAAs into the fluorophore of GFP, altering its photophysical properties.     

Prospects for lanthanides in structural biology by NMR

The advent of different lanthanide-binding reagents has made site-specific labelling of proteins with paramagnetic lanthanides a viable proposition. This brings many powerful techniques originally established and demonstrated for paramagnetic metalloproteins into the mainstream of structural biology. The promise is that, by exploiting the long-range effects of paramagnetism, lanthanide labelling will allow the study of larger proteins and protein-ligand complexes with greater ease and accuracy than hitherto possible. In particular, lanthanide-induced pseudocontact shifts (PCS) provide powerful restraints and 3D structure determination using PCS as the only source of experimental restraints will probably be possible with data obtained from samples with different lanthanide-tagging sites. Cell-free protein synthesis is positioned to play an important role in this strategy, as an inexpensive source of selectively labelled protein samples and for easy site-specific incorporation of unnatural lanthanide-binding amino acids.     

Direct protein–protein conjugation by genetically introducing bioorthogonal functional groups into proteins

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein–protein conjugates has gained research interest. In this study, site-specific protein–protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein–protein conjugates and protein complexes of biochemical significance.     

Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins

Modern drug discovery strongly depends on the availability of target proteins in sufficient amounts and with desired properties. For some applications, proteins have to be produced with specific modifications such as tags for protein purification, fluorescent or radiometric labels for detection, glycosylation and phosphorylation for biological activity, and many more. It is well known that covalent modifications can have adverse effects on the biological activity of some target proteins. It is therefore one of the major challenges in protein chemistry to generate covalent modifications without affecting the biological activity of the target protein. Current procedures for modification mostly rely on non-specific labelling of lysine or cysteine residues on the protein of interest, but alternative approaches dedicated to site-specific protein modification are being developed and might replace most of the commonly used methodologies. In this study, we investigated two novel methods where target proteins can be expressed in E. coli with a fusion partner that allows protein modification in a covalent and highly selective manner. Firstly, we explored a method based on the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) as a fusion tag for site-directed attachment of small molecules. The AGT-tag (SNAP-tag) can accept almost any chemical moiety when it is attached to the guanine base through a benzyl group. In our experiments we were able to label a target protein fused to the AGT-tag with various fluorophores coupled to O6-benzylguanine. Secondly, we tested in vivo and in vitro site-directed biotinylation with two different tags, consisting of either 15 (AviTag) or 72 amino acids (BioEase tag), which serve as a substrate for bacterial biotin ligase birA. When birA protein was co-expressed in E. coli biotin was incorporated almost completely into a model protein which carried these recognition tags at its C-terminus. The same findings were also obtained with in vitro biotinylation assays using pure birA independently over-expressed in E. coli and added to the biotinylation reaction in the test tube. For both biotinylation methods, peptide mapping and LC-MS proved the highly site-specific modification of the corresponding tags. Our results indicate that these novel site-specific labelling reactions work in a highly efficient manner, allow almost quantitative labelling of the target proteins, have no deleterious effect on the biological activity and are easy to perform in standard laboratories.     

An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media

The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR field. Here, we describe a novel protocol for incorporating unnatural amino acids into fully deuterated proteins using glucose-based media (which are relevant to the production, for example, of amino acid-specific methyl-labeled proteins used in the study of large molecular weight systems). The method consists of pre-induction of the pEVOL plasmid encoding the tRNA/aminoacyl-tRNA synthetase pair in a rich, H₂O-based medium prior to exchanging the culture into a D₂O-based medium. Our protocol results in high level of isotopic incorporation (~95%) and retains the high expression level of the target protein observed in Luria–Bertani medium.     

Site-specific one-pot dual labeling of DNA by orthogonal cycloaddition chemistry

Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.