Incorporation Of Unnatural Amino Acids Research Articles

Site-specific antibody fragment conjugates for targeted imaging.

This work details the use of amber suppression-mediated genetic incorporation of unnatural amino acids (UAAs), specifically p-azido-l-phenylalanine (pAzF) and p-acetyl-l-phenylalanine (pAcF), to develop site-specifically labeled antibody Fab fragments. These antibody fragment conjugates represent a novel class of imaging agents with optimal stability, efficacy, and pharmacological properties, which have demonstrated promising potential for probing and understanding the in vivo bio-distributions of protein targets of interest. This chapter provides general guidelines for preparing these Fab conjugates, and details of follow-up bioassays such as single-agent based positron emission tomography (PET) imaging of immune-checkpoint protein PD-L1, and the use of GCN4-mediated switchable antibody conjugates for near-infrared fluorescent imaging of cancer-related biomarkers.

Characterization of a hyperphosphorylated variant of G protein-coupled receptor kinase 5 expressed in E. coli

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK subfamilies that does not need post-translational lipidation or other binding partners to exhibit full activity against GPCRs, rendering it a useful tool for biophysical studies directed at characterizing GRK function. However, recombinant expression of GRK5 has thus far been limited to insect and mammalian systems. Here, we describe the expression of functional GRK5 in E. coli and its purification and biochemical characterization. Bacterially expressed GRK5 is hyperphosphorylated, primarily in regions known to be flexible from prior crystal structures, which slightly decreases its catalytic activity toward receptor substrates. Mutation of a single phosphorylation site, Thr10, restores kinetic parameters to those of GRK5 purified from insect cells. Consequently, bacterial expression will allow for production of GRK5 at a reduced cost and faster pace and would facilitate production of isotopically labeled kinase for NMR studies or for the incorporation of unnatural amino acids.

Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, the utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. Besides, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which could not be achieved in a living cell.

Miklós Bodanszky Award Lecture: Selective chalcogen chemistry to study protein science.

In recent decades, chemical protein synthesis and the development of chemoselective reactions-including ligation reactions-have led to significant breakthroughs in protein science. Among them are a better understanding of protein structure-function relationships, the study of protein posttranslational modifications, exploration of protein design, unnatural amino acid incorporation, and the study of therapeutic proteins and protein folding. Chalcogen chemistry, especially that of sulfur and selenium, is quite rich, and we have witnessed continuous progress in this field in recent years. In this short review, we will instead summarize three stories that we have recently presented on chalcogen chemistry and its impact on protein science, which was presented in the Miklós Bodanszky Award Lecture at the 35th European Peptide Society Meeting in Dublin, Ireland, 26 August 2018.

Recent Advances in Enzyme Engineering through Incorporation of Unnatural Amino Acids

The development of new enzyme engineering technologies has been actively pursued as the industrial use of biocatalysts is rapidly increasing. Traditional enzyme engineering has been limited to changing the functional properties of enzymes by replacing one amino acid with the other 19 natural amino acids. However, the incorporation of unnatural amino acids (UAAs) has been exploited to manipulate efficient enzymes for biocatalysis. This has been an effective enzyme engineering technique by complementing and extending the limits of traditional enzymatic functional changes. This review paper describes the basic functions of the new functional groups of UAAs used in enzyme engineering and the utilization of UAAs in the formation of chemical bonds in the proteins. The recent developments of UAA-mediated enzymology and its applicability in industry, pharmaceutical and other research areas to overcome the limitations of existing enzymes is also emphasized.

Site-Specific Cross-Linking of a p19 Viral Suppressor of RNA Silencing Protein and Its RNA Targets Using an Expanded Genetic Code.

The p19 viral suppressor of RNA silencing protein has useful applications in biotechnology due to its high affinity for binding to small RNAs such as small interfering RNAs (siRNAs). Also, its applications for the study and modulation of microRNAs are actively expanding. Here we demonstrate the successful site-specific incorporation of a photoactivatable unnatural amino acid, p-azido-l-phenylalanine (AzF), for cross-linking to RNA substrates into the p19 sequence. Incorporation of AzF was performed at three positions in the protein near the RNA binding site: K67, R115, and T111. Incorporation of AzF at position T111 of p19 did not affect the binding affinity of p19 for siRNAs and also showed nanomolar affinity for human microRNA miR-122. The affinity was less favorable with AzF incorporation at two other positions, suggesting the sensitivity of placement of the unnatural amino acid. Exposure of the T111AzF in complex with either siRNA or miRNA to ultraviolet light resulted in cross-linking of the protein with the RNA, but no cross-linking could be detected with the wild-type protein. Our results demonstrate that p19-T111AzF can be used for detection of small RNAs, including human miR-122, with high sensitivity and to irreversibly sequester these RNAs through covalent photo-cross-linking.

Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement.

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

Sensitive detection of type G botulinum neurotoxin through Endopep-MS peptide substrate optimization

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A–G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD50 toxin present in buffer solution.

Advances and Challenges in Cell-Free Incorporation of Unnatural Amino Acids Into Proteins.

Incorporation of unnatural amino acids (UNAAs) into proteins currently is an active biological research area for various fundamental and applied science. In this context, cell-free synthetic biology (CFSB) has been developed and recognized as a robust testing and biomanufacturing platform for highly efficient UNAA incorporation. It enables the orchestration of unnatural biological machinery toward an exclusive user-defined objective of unnatural protein synthesis. This review aims to overview the principles of cell-free unnatural protein synthesis (CFUPS) systems, their advantages, different UNAA incorporation approaches, and recent achievements. These have catalyzed cutting-edge research and diverse emerging applications. Especially, present challenges and future trends are focused and discussed. With the development of CFSB and the fusion with other advanced next-generation technologies, CFUPS systems would explicitly deliver their values for biopharmaceutical applications.

Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine.

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.

Using genetically incorporated unnatural amino acids to control protein functions in mammalian 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.

Biogenesis, quality control, and structural dynamics of proteins as explored in living cells via site-directed photocrosslinking.

Protein biogenesis and quality control are essential to maintaining a functional pool of proteins and involve numerous protein factors that dynamically and transiently interact with each other and with the substrate proteins in living cells. Conventional methods are hardly effective for studying dynamic, transient, and weak protein-protein interactions that occur in cells. Herein, we review how the site-directed photocrosslinking approach, which relies on the genetic incorporation of a photoreactive unnatural amino acid into a protein of interest at selected individual amino acid residue positions and the covalent trapping of the interacting proteins upon ultraviolent irradiation, has become a highly efficient way to explore the aspects of protein contacts in living cells. For example, in the past decade, this approach has allowed the profiling of the in vivo substrate proteins of chaperones or proteases under both physiologically optimal and stressful (e.g., acidic) conditions, mapping residues located at protein interfaces, identifying new protein factors involved in the biogenesis of membrane proteins, trapping transiently formed protein complexes, and snapshotting different structural states of a protein. We anticipate that the site-directed photocrosslinking approach will play a fundamental role in dissecting the detailed mechanisms of protein biogenesis, quality control, and dynamics in the future.

Site-Specific Labeling of Proteins Using Unnatural Amino Acids.

Labeling of a protein with a specific dye or tag at defined positions is a critical step in tracing the subtle behavior of the protein and assessing its cellular function. Over the last decade, many strategies have been developed to achieve selective labeling of proteins in living cells. In particular, the site-specific unnatural amino acid (UAA) incorporation technique has gained increasing attention since it enables attachment of various organic probes to a specific position of a protein in a more precise way. In this review, we describe how the UAA incorporation technique has expanded our ability to achieve site-specific labeling and visualization of target proteins for functional analyses in live cells.

Construction of Bacterial Cells with an Active Transport System for Unnatural Amino Acids.

Engineered organisms with an expanded genetic code have attracted much attention in chemical and synthetic biology research. In this work, engineered bacterial organisms with enhanced unnatural amino acid (UAA) uptake abilities were developed by screening periplasmic binding protein (PBP) mutants for recognition of UAAs. A FRET-based assay was used to identify a mutant PBP (LBP-AEL) with excellent binding affinity ( Kd ≈ 500 nM) to multiple UAAs from 37 mutants. Bacterial cells expressing LBP-AEL showed up to 5-fold enhanced uptake of UAAs, which was determined by genetic incorporation of UAAs into a green fluorescent protein and measuring UAA concentration in cell lysates. To the best of our knowledge, this work is the first report of engineering cellular uptake of UAAs and could provide an impetus for designing advanced unnatural organisms with an expanded genetic code, which function with the efficiency comparable to that of natural organisms. The system would be useful to increase mutant protein yield from lower concentrations of UAAs for industrial and large-scale applications. In addition, the techniques used in this report such as the sensor design and the measurement of UAA concentration in cell lysates could be useful for other biochemical applications.

Incorporation of Unnatural Amino Acids via Proteomically Informed Selective Pressure Techniques

Determining the three‐dimensional structure of a protein using x‐ray crystallography can be enhanced by complete or partial incorporation of unnatural amino acids into a target protein. Most naturally occurring atoms do not have sufficient mass to diffract electrons, therefore, heavy atom derivatives of target proteins are required. The Incorporation of unnatural metalloid containing amino acids provides a clear solution to the problem. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotroph E. coli DL41(DE3) (pCock) expression system. Both telluromethionine and methionine exposed cultures were analyzed with the objective of identifying differentially expressed proteins. The analysis was carried out using 2D separation with isoelectric focusing serving in the first dimension and SDS polyacrylamide gel electrophoresis in the second. In‐gel tryptic digestion followed by peptide mass fingerprinting using nano‐ESI/qTOF/MS allowed identification of differentially expressed proteins. A few of the differentially expressed proteins identified as down‐regulated in systems exposed to TeMet were UPase, ZnuA, phosphoserine aminotransferase, and fructose‐bisphosphate aldolase. Understanding the biological roles of differentially expressed proteins was used to improve methods of TeMet bioincorporation using the selective pressure technique.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessing Cell Growth in a Microtiter Plate Reader with Selective Pressure Incorporation of Unnatural Selenium and Tellurium Containing Amino Acids

Metalloid‐containing unnatural amino acids, when incorporated into target proteins, provide a means of solving for three‐dimensional structure by MAD or SAD techniques. The use of Tellurium‐containing amino acids are of special interest since they can also be exploited by more traditional MIR techniques, which don't require travel to synchrotron centers. Tellurium, when incorporated into protein as telluromethionine, generally induces toxicity to E. coli. Selenium, when incorporated as selenomethionine, induces much lower toxic effects to cell culture but remains problematic, at times. Altering growth conditions can potentially reduce such toxicity. This study demonstrates a rapid screening method for biosynthetic incorporation of unnatural amino acids by various forms of selective pressure.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Crystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway.

Photoactivatable fluorescent proteins (PA-FPs) are a powerful non-invasive tool in high-resolution live-cell imaging. They can be converted from an inactive to an active form by light, enabling the spatial and temporal trafficking of proteins and cell dynamics. PA-FPs have been previously generated by mutating selected residues in the chromophore or in its close proximity. A new strategy to generate PA-FPs is the genetic incorporation of unnatural amino acids (UAAs) containing photocaged groups using unique suppressor tRNA/aminoacyl-tRNA synthetase pairs. We set out to develop a photoactivatable GFP variant suitable for time-resolved structural studies. Here, we report the crystal structure of superfolder GFP (sfGFP) containing the UAA ortho-nitrobenzyl-tyrosine (ONBY) at position 66 and its spectroscopic characterization. Surprisingly, the crystal structure (to 2.7Å resolution) reveals a dimeric domain-swapped arrangement of sfGFP66ONBY with residues 1-142 of one molecule associating with residues 148-234 from another molecule. This unusual domain-swapped structure supports a previously postulated GFP folding pathway that proceeds via an equilibrium intermediate.

Site-Specific Immuno-PET Tracer to Image PD-L1.

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', N″-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, 64Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA.

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a Kd value of 410 nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.

Expansion of Redox Chemistry in Designer Metalloenzymes.

Many artificial enzymes that catalyze redox reactions have important energy, environmental, and medical applications. Native metalloenzymes use a set of redox-active amino acids and cofactors as redox centers, with a potential range between -700 and +800 mV versus standard hydrogen electrode (SHE, all reduction potentials are versus SHE). The redox potentials and the orientation of redox centers in native metalloproteins are optimal for their redox chemistry. However, the limited number and potential range of native redox centers challenge the design and optimization of novel redox chemistry in metalloenzymes. Artificial metalloenzymes use non-native redox centers and could go far beyond the natural range of redox potentials for novel redox chemistry. In addition to designing protein monomers, strategies for increasing the electron transfer rate in self-assembled protein complexes and protein-electrode or -nanomaterial interfaces will be discussed. Redox reactions in proteins occur on redox active amino acid residues (Tyr, Trp, Met, Cys, etc.) and cofactors (iron sulfur clusters, flavin, heme, etc.). The redox potential of these redox centers cover a ∼1.5 V range and is optimized for their specific functions. Despite recent progress, tuning the redox potential for amino acid residues or cofactors remains challenging. Many redox-active unnatural amino acids (UAAs) can be incorporated into protein via genetic codon expansion. Their redox potentials extend the range of physiologically relevant potentials. Indeed, installing new redox cofactors with fined-tuned redox potentials is essential for designing novel redox enzymes. By combining UAA and redox cofactor incorporation, we harnessed light energy to reduce CO2 in a fluorescent protein, mimicking photosynthetic apparatus in nature. Manipulating the position and reduction potential of redox centers inside proteins is important for optimizing the electron transfer rate and the activity of artificial enzymes. Learning from the native electron transfer complex, protein-protein interactions can be enhanced by increasing the electrostatic interaction between proteins. An artificial oxidase showed close to native enzyme activity with optimized interaction with electron transfer partner and increased electron transfer efficiency. In addition to the de novo design of protein-protein interaction, protein self-assembly methods using scaffolds, such as proliferating cell nuclear antigen, to efficiently anchor enzymes and their redox partners. The self-assembly process enhances electron transfer efficiency and enzyme activity by bringing redox centers into close proximity of each other. In addition to protein self-assembly, protein-electrode or protein-nanomaterial self-assembly can also promote efficient electron transfer from inorganic materials to enzyme active sites. Such hybrid systems combine the efficiency of enzyme reactions and the robustness of electrodes or nanomaterials, often with advantageous catalytic activities. By combining these strategies, we can not only mimic some of nature's most fascinating reactions, such as photosynthesis and aerobic respiration, but also transcend nature toward environmental, energy, and health applications.