Unnatural Amino Acid Mutagenesis Research Articles

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the vertebrate nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type iGluRs using genetically encoded unnatural amino acid (UAA) photocross-linkers, p-benzoyl-l-phenylalanine (BzF) and p-azido-l-phenylalanine (AzF). We introduced these UAAs at sites throughout the TMD of the GluA2 receptor and characterized the mutants in patch-clamp recordings, exposing them to glutamate and ultraviolet (UV) light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence for an interaction between the Pre-M1 and the M4 TMD helix during desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter in the M2 segment, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric ion channels, selectivity filter gating.

Membrane-Permeant, Environment-Sensitive Dyes Generate Biosensors within Living Cells.

Dyes with environment-sensitive fluorescence have proven useful to study the spatiotemporal dynamics of protein activity in living cells. When attached to proteins, their fluorescence can reflect protein conformational changes, post-translational modifications, or protein interactions. However, the utility of such dye-protein conjugates has been limited because it is difficult to load them into cells. They usually must be introduced using techniques that perturb cell physiology, limit throughput, or generate fluorescent vesicles (e.g., electroporation, microinjection, or membrane transduction peptides). Here we circumvent these problems by modifying a proven, environment-sensitive biosensor fluorophore so that it can pass through cell membranes without staining intracellular compartments and can be attached to proteins within living cells using unnatural amino acid (UAA) mutagenesis. Reactive groups were incorporated for attachment to UAAs or small molecules (mero166, azide; mero167, alkyne; mero76, carboxylic acid). These dyes are bright and fluoresce at long wavelengths (reaching ε = 100 000 M-1 cm-1, ϕ = 0.24, with excitation 565 nm and emission 594 nm). The utility of mero166 was demonstrated by in-cell labeling of a UAA to generate a biosensor for the small GTPase Cdc42. In addition, conjugation of mero166 to a small molecule produced a membrane-permeable probe that reported the localization of the DNA methyltransferase G9a in cells. This approach provides a strategy to access biosensors for many targets and to more practically harness the varied environmental sensitivities of synthetic dyes.

Labeling Proteins at Site-Specifically Incorporated 5-Hydroxytryptophan Residues Using a Chemoselective Rapid Azo-Coupling Reaction.

Chemoselective protein labeling is a valuable tool in the arsenal of modern chemical biology. The unnatural amino acid mutagenesis technology provides a powerful way to site-specifically introduce nonnatural chemical functionalities into recombinant proteins, which can be subsequently functionalized in a chemoselective manner. Even though several strategies currently exist to selectively label recombinant proteins in this manner, there is considerable interest for the development of additional chemoselective reactions that are fast, catalyst-free, use readily available reagents, and are compatible with existing conjugation chemistries. Here we describe a method to express recombinant proteins in E. coli site-specifically incorporating 5-hydroxytryptophan, followed by the chemoselective labeling of this residue using a chemoselective rapid azo-coupling reaction.

Genetic Code Expansion in Animals.

Expanding the genetic code to enable the incorporation of unnatural amino acids into proteins in biological systems provides a powerful tool for studying protein structure and function. While this technology has been mostly developed and applied in bacterial and mammalian cells, it recently expanded into animals, including worms, fruit flies, zebrafish, and mice. In this review, we highlight recent advances toward the methodology development of genetic code expansion in animal model organisms. We further illustrate the applications, including proteomic labeling in fruit flies and mice and optical control of protein function in mice and zebrafish. We summarize the challenges of unnatural amino acid mutagenesis in animals and the promising directions toward broad application of this emerging technology.

Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes

The bacteria-derived tyrosyl-tRNA synthetase (TyrRS)/tRNA pair was first used for unnatural amino acid (Uaa) mutagenesis in eukaryotic cells over 15years ago. It provides an ideal platform to genetically encode numerous useful Uaas in eukaryotes. However, this pair has been engineered to charge only a small collection of Uaas to date. Development of Uaa-selective variants of this pair has been limited by technical challenges associated with a yeast-based directed evolution platform, which is currently required to alter its substrate specificity. Here we overcome this limitation by enabling its directed evolution in an engineered strain of E.coli (ATMY), where the endogenous TyrRS/tRNA pair has been functionally replaced with an archaeal counterpart. The facile E.coli-based selection system enabled rapid engineering of this pair to develop variants that selectively incorporate various Uaas, including p-boronophenylalanine, into proteins expressed in mammalian cells as well as in the ATMY strain of E.coli.

Photoaffinity Cross-Linking and Unnatural Amino Acid Mutagenesis Reveal Insights into Calcitonin Gene-Related Peptide Binding to the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1) Complex.

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

Controlling the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells.

Large efforts have been devoted to genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells. Intriguingly, the trend of quadruplet codon decoding efficiency among the tested tRNA variants in mammalian cells was largely the same as that in E.coli. We subsequently demonstrate the utility of quadruplet codon decoding by the construction of the first HIV-1 mutant that lacks any in-frame amber nonsense codons and can be precisely activated by the decoding of a genomically embedded UAGA codon with an unnatural amino acid. Such conditionally activatable HIV-1 mutant can likely facilitate both fundamental investigations of HIV-1 as well as vaccine developments. The use of quadruplet codon, instead of an amber nonsense codon, to control HIV-1 replication has the advantage in that the correction of a frameshift caused by a quadruplet codon is much less likely than the reversion of an amber codon back into a sense codon in HIV-1.

Investigation of Trimethyllysine Binding by the HP1 Chromodomain via Unnatural Amino Acid Mutagenesis.

Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation-π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation-π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation-π-mediated Kme3 recognition. Although cation-π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation-π interactions.

Site-Directed Unnatural Amino Acid Mutagenesis to Investigate Potassium Channel Pharmacology in Xenopus laevis Oocytes.

Unnatural amino acid mutagenesis is a useful tool enabling detailed investigation of ion channel structure-function relationships and pharmacology. Methods have been developed to apply this technique to different heterologous systems for electrophysiological studies, with each system offering unique advantages and limitations. Synthesis of aminoacylated-tRNA followed by injection into Xenopus laevis oocytes is beneficial because it allows for the incorporation of a wide range of unnatural sidechains, including amino acids with subtle structural differences. Here, we describe a protocol for unnatural amino acid mutagenesis implemented in our lab to study the pharmacology of KCNQ voltage-gated potassium channel opener compounds. This protocol should be applicable to other ion channels and receptor types amenable for functional studies in Xenopus laevis oocytes.

Fluorescent Protein-Based Turn-On Probe through a General Protection-Deprotection Design Strategy.

We demonstrated a general protection-deprotection strategy for the design of fluorescent protein biosensors through the construction of a turn-on Hg2+ sensor. A combination of fluorescent protein engineering and unnatural amino acid mutagenesis was used. Unlike previously reported fluorescent protein-based Hg2+ sensors that relied on the binding of Hg2+ to the sulfhydryl group of cysteine residues, a well-established chemical reaction, oxymercuration, was transformed into biological format and incorporated into our sensor design. This novel Hg2+ sensor displayed good sensitivity and selectivity both in vitro and in live bacterial cells. Over 60-fold change in fluorescence signal output was observed in the presence of 10 μM Hg2+, while such a change was undetectable when nine other metal ions were tested. This new design strategy could expand the repertoire of fluorescent protein-based biosensors for the detection of small-molecule analytes.

Expanding the genetic code of mammalian cells

In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology.

Discovery of a Small-Molecule Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28.

MicroRNAs (miRNAs) regulate gene expression by targeting protein-coding transcripts that are involved in various cellular processes. Thus, miRNA biogenesis has been recognized as a novel therapeutic target. Especially, the let-7 miRNA family is well-known for its tumor suppressor functions and is downregulated in many cancer cells. Lin28 protein binds to let-7 miRNA precursors to inhibit their maturation. Herein, we developed a FRET-based, high-throughput screening system to identify small-molecule inhibitors of the Lin28-let-7 interaction. We employed unnatural amino acid mutagenesis and bioorthogonal chemistry for the site-specific fluorescent labeling of Lin28, which ensures the robustness and reliability of the FRET-based protein-miRNA binding assay. Using this direct binding assay, we identified an inhibitor of the oncogenic Lin28-let-7 interaction. The inhibitor enhanced the production of let-7 miRNAs in Lin28-expressing cancer cells and reduced the level of let-7 target oncogene products.

Radical protein modifications

Researchers have devised a simple method called chemical mutagenesis that modifies proteins at specific sites. It is the first general technique for creating new C–C bond linkages in proteins and allows chemists to add a wide range of modifying groups: new sidechains, including unnatural ones; so-called posttranslational modifications that cells normally add enzymatically, including methyl groups and phosphates; and individual or chains of sugars (Science 2016, DOI: 10.1126/science.aag1465). The approach, developed by Benjamin G. Davis and coworkers at the University of Oxford, could make it easier to study the structure and biological functions of proteins—especially when combined with other protein-modifying techniques such as site-directed mutagenesis, unnatural amino acid mutagenesis, and bioorthogonal chemistry. It could also lead to new types of protein drugs or “synthetic biologics.” Modifying proteins by creating new C–C bonds has been largely out of reach. Most posttranslational modifications cre...

Virus-Enabled Optimization and Delivery of the Genetic Machinery for Efficient Unnatural Amino Acid Mutagenesis in Mammalian Cells and Tissues.

Unnatural amino acid (UAA) mutagenesis of recombinant proteins in live mammalian cells requires coexpression of the mutant target, as well as an engineered tRNA/aminoacyl-tRNA synthetase pair. The ability to readily determine the optimal relative expression levels of these three genetic components for efficient expression of the UAA-modified target is highly desirable, but remains challenging to accomplish. Here we report a facile strategy to achieve this by taking advantage of the efficient gene-delivery by a baculovirus vector, which enables systematic variation of the expression level of each genetic component in a population-wide manner. Insights gained from this study led to the design of an optimal expression system, which can be delivered into mammalian cells by a single baculovirus vector to provide significantly improved UAA incorporation efficiency at a low virus load. Furthermore, this optimized baculovirus vector was shown to enable efficient UAA mutagenesis of proteins expressed in mouse brain tissue.

In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures.

Chemically self-assembled nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation, and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly into CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging, and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR(2)) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[(64)Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN's binding affinity toward U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of (64)Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an in vivo profile between those of rapidly clearing small molecules and slow clearing antibodies. Thus, CSANs offer a modular, programmable, and stable protein based platform that can be used for in vivo drug delivery and imaging applications.

Using Unnatural Amino Acids to Probe the Molecular Basis for Herg Drug Block

The human Ether-a-go-go Related Gene (hERG) voltage gated potassium channel underlies the rapid delayed rectifier current, IKr, which plays a central role in cardiac action potential repolarization. Inhibition of IKr results in a heart rhythm disorder, long QT syndrome type 2, which can lead to torsades de pointes and sudden death. Unfortunately, hERG is blocked by a wide range of therapeutic drug classes and this has led federal drug regulators to mandate hERG-block screening as a requirement in drug development. Past mutagenesis studies have highlighted two aromatic residues, Y652 and F656, in the pore region of hERG as crucial for high affinity drug block of the channel [1]. The molecular basis for drug interaction with these sites is often suggested to be via cation-pi interactions. We have directly tested this hypothesis using unnatural amino acid mutagenesis to incorporate fluorinated phenylalanine derivatives at Y652 and F656 in Xenopus laevis oocytes. Unnatural side chains can be efficiently incorporated into hERG at both positions tested, with very little non-specific incorporation. Using two-electrode voltage clamp recordings, we measured the affinity of known hERG blockers terfenadine and quinidine on WT and fluoro-Phe substituted channels. The effects of fluorination are not consistent with a cation-pi interaction as the primary force governing the action of these drugs. However, our findings demonstrate the feasibility of the nonsense suppression approach in hERG, and pave the way for detailed dissection of the chemical basis of hERG blockade.1. Mitcheson, J.S., et al., A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci U S A, 2000. 97(22): p. 12329-33.

Clickable silk: novel silk materials containing azide reactive groups

Event Abstract Back to Event Clickable silk: novel silk materials containing azide reactive groups Hidetoshi Teramoto1, Mizuki Sasaki2, Yasushi Tamada2, Kenichi Nakajima1 and Katsura Kojima1* 1 National Institute of Agrobiological Sciences, Genetically Modified Organism Research Center, Japan 2 Shinshu University, Faculty of Textile Science and Technology, Japan Introduction: Silk fibroin protein from the domesticated silkworms, B. mori, has been studied as a candidate for biomaterials development due of its mechanical toughness, biocompatibility, and biodegradability[1]. In this study, a rapid, easy, and flexible method to modify silk fibroin was developed based on the unnatural amino acid mutagenesis methodology of proteins[2]. We previously succeeded in incorporating an unnatural amino acid, 4-azidophenylalanine (AzPhe) (Fig. 1), into silk fibroin by expressing a mutant of phenylalanyl-tRNA synthetase (PheRS) with expanded amino acid recognition capacity in silk glands[3]. The incorporation of AzPhe has made silk fibroin “clickable”: desired functional molecules could be attached to it by azide-specific click reactions[2]. In this paper, the fabrication of materials from clickable silk and their functionalization by click reactions were investigated. Photo-patternability of clickable silk materials was also investigated. Materials and Methods: Fig. 1 illustrates the preparation of clickable silk materials: AzPhe mixed in a commercially-available standard diet was administered to transgenic silkworms (5th instar) until they started to spin cocoons. The harvested cocoons were then processed into threads, films, and porous sponges with established techniques[4]. Results and Discussion: Clickable silk materials (threads, films, and porous sponges) were subjected to click reactions with carboxyrhodamine 110 alkyne (green fluorescence) or sulforhodamine B DBCO (red fluorescence) (Fig. 2). In any forms, the clickable silk materials were distinctly modified by the fluorescent molecules, thus demonstrating the high specificity of click reactions toward azide groups. We attributed the weak fluorescence observed for normal silk materials to non-specific binding of the reagents. Since azide groups in AzPhe are known to exhibit photolysis upon UV irradiation, we expected that the reactivity of azide groups would be geometrically controlled on clickable silk materials. Clickable silk films (cut to 7 × 7 mm2), irradiated with 365 nm UV light for 1 h through micropatterned photomasks, were reacted with carboxyrhodamine 110 alkyne (green fluorescence) or sulforhodamine B DBCO (red fluorescence) (Fig. 3). The micropatterns on the photomasks were successfully transferred to clickable silk films, demonstrating that desired functional molecules could be directly patterned onto clickable silk materials on a micrometer scale. Conclusions: Clickable silk materials with photo-patternability were created by the incorporation of an unnatural amino acid, AzPhe, into silk fibroin. They were easily modified by click reactions, suggesting that they could be a versatile platform on which to produce functionalized biomaterials for wide applications. This study also demonstrates the potential of the unnatural amino acid mutagenesis of proteins to generate novel types of protein-based biomaterials. This work was supported by JSPS KAKENHI Grant Numbers 24688008,15K07800.

Atomic basis for therapeutic activation of neuronal potassium channels.

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2–5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

ChemInform Abstract: Semi‐Synthesis of Thioamide Containing Proteins

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Atom‐by‐atom engineering of voltage‐gated ion channels: Magnified insights into function and pharmacology

Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino acids into proteins. We also review insights into the function and pharmacology of voltage-gated ion channels that have emerged from unnatural amino acid mutagenesis approaches.