Incorporation Of Unnatural Amino Acids Research Articles

Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis.

The hydrogen bonding network that surrounds the flavin in blue light using flavin adenine dinucleotide (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase from Oscillatoria acuminata (OaPAC). Our work elucidates the direct connection between BLUF photoactivation and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate the formation of the neutral flavin radical as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogues to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FAD•-. The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the adenylyl cyclase (AC) domain. While the 3F-Y6 and 2,3-F2Y6 variants undergo the complete photocycle and catalyze the conversion of ATP into cAMP, enzyme activity is abolished in the 3,5-F2Y6 and 2,3,5-F3Y6 variants where the photocycle is halted at FAD•-. Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in AC activity.

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

Efficient In Vitro Full-Sense-Codons Protein Synthesis.

Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs are screened for efficient all stop-codons decoding to construct a codon-dependent termination defect in vitro protein synthesis with all 64 sense-codons, iPSSC. Finally, this full sense genetic code achieves significant improvement in the incorporation of distinct unnatural amino acids at up to 12 positions and synthesis of protein encoding consecutive NNN codons. By decoding all information in nucleotides to amino acids, iPSSC may hold great potential in building artificial protein synthesis beyond the cell.

Stereocontrolled 1,3-nitrogen migration to access chiral α-amino acids.

α-Amino acids are essential for life as building blocks of proteins and components of diverse natural molecules. In both industry and academia, the incorporation of unnatural amino acids is often desirable for modulating chemical, physical, and pharmaceutical properties. We here report a protocol for the economical and practical synthesis of optically active α-amino acids based on an unprecedented stereocontrolled 1,3-nitrogen shift. Our method employs abundant and easily accessible carboxylic acids as starting materials, which are first connected to a nitrogenation reagent, followed by a highly regio- and enantioselective ruthenium- or iron-catalyzed C(sp3)−H amination. This straightforward method displays a very broad scope, providing rapid access to optically active α-amino acids with aryl, allyl, propargyl, and alkyl side chains, and also permits stereocontrolled late-stage amination of carboxylic acid-containing drugs and natural products.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1–6) and all of them share highly conserved six transmembrane (TM) domains (S1–S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1–S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1–S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1–S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1–S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Genetic code expansion and its application in characterization and imaging of proteins

Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.

A Chemical Biology Primer for NMR Spectroscopists

Among structural biology techniques, NMR spectroscopy offers unique capabilities that enable the atomic resolution studies of dynamic and heterogeneous biological systems under physiological and native conditions. Complex biological systems, however, often challenge NMR spectroscopists with their low sensitivity, crowded spectra or large linewidths that reflect their intricate interaction patterns and dynamics. While some of these challenges can be overcome with the development of new spectroscopic approaches, chemical biology can also offer elegant and efficient solutions at the sample preparation stage. In this tutorial, we aim to present several chemical biology tools that enable the preparation of selectively and segmentally labeled protein samples, as well as the introduction of site-specific spectroscopic probes and post-translational modifications. The four tools covered here, namely cysteine chemistry, inteins, native chemical ligation, and unnatural amino acid incorporation, have been developed and optimized in recent years to be more efficient and applicable to a wider range of proteins than ever before. We briefly introduce each tool, describe its advantages and disadvantages in the context of NMR experiments, and offer practical advice for sample preparation and analysis. We hope that this tutorial will introduce beginning researchers in the field to the possibilities chemical biology can offer to NMR spectroscopists, and that it will inspire new and exciting applications in the quest to understand protein function in health and disease.

Incorporation of metal-chelating unnatural amino acids into halotag for allylic deamination

The potential of artificial metalloenzymes has led to an increase in interest for the design of novel metal-binding sites in proteins. Metal-chelating unnatural amino acids offer an auspicious solution to engineer active metal sites in a defined way. Herein, we describe the introduction of four metal-chelating unnatural amino acids into HaloTag, an attractive scaffold for the assembly of functional artificial metalloenzymes. HaloTag, engineered with 2-amino-3-(8-hydroxyquinolin-5-yl)propanoic acid (HQ-Ala-1) was used to assemble an artificial metalloenzyme for improved allylic deamination upon complementation with [(η5-C5H5)Ru(MeCN)3]+.

A Simplified Protocol to Incorporate the Fluorescent Unnatural Amino Acid ANAP into Xenopus laevis Oocyte-Expressed P2X7 Receptors.

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein.

Recombinant and genetic code expanded collagen-like protein as a tailorable biomaterial.

Collagen occurs in nature with a dedicated triple helix structure and is the most preferred biomaterial in commercialized medical products. However, concerns on purity, disease transmission, and the reproducibility of animal derived collagen restrict its applications and warrants alternate recombinant sources. The expression of recombinant collagen in different prokaryotic and eukaryotic hosts has been reported with varying degrees of success, however, it is vital to elucidate the structural and biological characteristics of natural collagen. The recombinant production of biologically functional collagen is restricted by its high molecular weight and post-translational modification (PTM), especially the hydroxylation of proline to hydroxyproline. Hydroxyproline plays a key role in the structural stability and higher order self-assembly to form fibrillar matrices. Advancements in synthetic biology and recombinant technology are being explored for improving the yield and biomimicry of recombinant collagen. It emerges as reliable, sustainable source of collagen, promises tailorable properties and thereby custom-made protein biomaterials. Remarkably, the evolutionary existence of collagen-like proteins (CLPs) has been identified in single-cell organisms. Interestingly, CLPs exhibit remarkable ability to form stable triple helical structures similar to animal collagen and have gained increasing attention. Strategies to expand the genetic code of CLPs through the incorporation of unnatural amino acids promise the synthesis of highly tunable next-generation triple helical proteins required for the fabrication of smart biomaterials. The review outlines the importance of collagen, sources and diversification, and animal and recombinant collagen-based biomaterials and highlights the limitations of the existing collagen sources. The emphasis on genetic code expanded tailorable CLPs as the most sought alternate for the production of functional collagen and its advantages as translatable biomaterials has been highlighted.

Cell-free synthetic biology: Orchestrating the machinery for biomolecular engineering

Due to inherent complexity, incompatibility, and variability in living cell systems, biomolecular engineering faces significant obstacles. To find novel solutions to these issues, researchers have turned to cell-free synthetic biology (CFSB), a relatively young field of study. Biochemical processes can be triggered in vitro through cell-free synthesis, providing a wider range of options for biomolecular engineering. Here, we provide a survey of recent advances in cell-free synthesis. These have sparked innovative studies in areas including the synthesis of complex proteins, incorporation of unnatural amino acids, precise post-translational modifications, high-throughput workflow, and synthetic biomolecular network regulation. CFSB has transformed the studies of biological machinery in a profound and practical way for versatile biomolecular engineering applications.

Rational incorporation of any unnatural amino acid into proteins by machine learning on existing experimental proofs

The unnatural amino acid (UAA) incorporation technique through genetic code expansion has been extensively used in protein engineering for the last two decades. Mutations into UAAs offer more dimensions to tune protein structures and functions. However, the huge library of optional UAAs and various circumstances of mutation sites on different proteins urge rational UAA incorporations guided by artificial intelligence. Here we collected existing experimental proofs of UAA-incorporated proteins in literature and established a database of known UAA substitution sites. By program designing and machine learning on the database, we showed that UAA incorporations into proteins are predictable by the observed evolutional, steric and physiochemical factors. Based on the predicted probability of successful UAA substitutions, we tested the model performance using literature-reported and freshly-designed experimental proofs, and demonstrated its potential in screening UAA-incorporated proteins. This work expands structure-based computational biology and virtual screening to UAA-incorporated proteins, and offers a useful tool to automate the rational design of proteins with any UAA.

Toward efficient multiple-site incorporation of unnatural amino acids using cell-free translation system

Amber suppression has been widely used to incorporate unnatural amino acids (UNAAs) with unique structures or functional side-chain groups into specific sites of the target protein, which expands the scope of protein-coding chemistry. However, this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein, which limits the development of unnatural proteins. To address this challenge, the suppression method using multiple termination codons (TAG, TAA or TGA) was proposed, and cell-free unnatural protein synthesis (CFUPS) system was employed. By the analysis of incorporating 3 different UNAAs (p-propargyloxy-l-phenylalanine, p-azyl-phenylalanine and L-4-Iodophenylalanine) and mass spectrometry, the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation. The CFUPS conditions were further optimized for better UNAA incorporation efficiency, including the orthogonal translation system (OTS) components, magnesium ions, and the redox environment. This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs, thereby synthesizing unnatural proteins with novel physicochemical functions.

Reagentless electrochemical biosensors through incorporation of unnatural amino acids on the protein structure

Typical protein biosensors employ chemical or genetic labeling of the protein, thus introducing an extraneous molecule to the wild-type parent protein, often changing the overall structure and properties of the protein. While these labeling methods have proven successful in many cases, they also have a series of disadvantages associated with their preparation and function. An alternative route for labeling proteins is the incorporation of unnatural amino acid (UAA) analogues, capable of acting as a label, into the structure of a protein. Such an approach, while changing the local microenvironment, poses less of a burden on the overall structure of the protein. L-DOPA is an analog of phenylalanine and contains a catechol moiety that participates in a quasi-reversible, two-electron redox process, thus making it suitable as an electrochemical label/reporter. The periplasmic glucose/galactose binding protein (GBP) was chosen to demonstrate this detection principle. Upon glucose binding, GBP undergoes a significant conformational change that is manifested as a change in the electrochemistry of L-DOPA. The electroactive GBP was immobilized onto gold nanoparticle-modified, polymerized caffeic acid, screen-printed carbon electrodes (GBP-LDOPA/AuNP/PCA/SPCE) for the purpose of direct measurement of glucose levels and serves as a proof-of-concept of the use of electrochemically-active unnatural amino acids as the label. The resulting reagentless GBP biosensors exhibited a highly selective and sensitive binding affinity for glucose in the micromolar range, laying the foundation for a new biosensing methodology based on global incorporation of an electroactive amino acid into the protein's primary sequence for highly selective electrochemical detection of compounds of interest.

Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction

Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.

Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids.

With increased attention to excellent biocatalysts, evolving methods based on nature or unnatural amino acid (UAAs) mutagenesis have become an important part of enzyme engineering. The emergence of powerful method through expanding the genetic code allows to incorporate UAAs with unique chemical functionalities into proteins, endowing proteins with more structural and functional features. To date, over 200 diverse UAAs have been incorporated site-specifically into proteins via this methodology and many of them have been widely exploited in the field of enzyme engineering, making this genetic code expansion approach possible to be a promising tool for modulating the properties of enzymes. In this context, we focus on how this robust method to specifically incorporate UAAs into proteins and summarize their applications in enzyme engineering for tuning and expanding the functional properties of enzymes. Meanwhile, we aim to discuss how the benefits can be achieved by using the genetically encoded UAAs. We hope that this method will become an integral part of the field of enzyme engineering in the future.

Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon.

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylSZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNApyl, such as pylSZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated.

A Robust Platform for Unnatural Amino Acid Mutagenesis in E. coli Using the Bacterial Tryptophanyl-tRNA synthetase/tRNA pair

We report the development of a robust user-friendly Escherichia coli (E. coli) expression system, derived from the BL21(DE3) strain, for site-specifically incorporating unnatural amino acids (UAAs) into proteins using engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS)-tRNATrp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNATrp pair in BL21(DE3) E. coli with an orthogonal counterpart from Saccharomyces cerevisiae, and reintroducing it into the resulting altered translational machinery tryptophanyl (ATMW-BL21) E. coli strain as an orthogonal nonsense suppressor. The resulting expression system benefits from the favorable characteristics of BL21(DE3) as an expression host, and is compatible with the broadly used T7-driven recombinant expression system. Furthermore, the vector expressing the nonsense-suppressing engineered EcTrpRS-tRNATrp pair was systematically optimized to significantly enhance the incorporation efficiency of various tryptophan analogs. Together, the improved strain and the optimized suppressor plasmids enable efficient UAA incorporation (up to 65% of wild-type levels) into several different proteins. This robust and user-friendly platform will significantly expand the scope of the genetically encoded tryptophan-derived UAAs.

Optimization of unnatural amino acid incorporation in collagen and the cross-linking through thioether bond

The source of recombinant collagen is clean, and it has the advantages of flexible sequence design, high yield and high purity, so it has a wide application prospect as biomaterials in tissue engineering and other fields. However, how to promote the cross-linking of recombinant collagen molecules and make them form a more stable spatial structure is the difficulty to be overcome in the design of recombinant collagen nanomaterials. Unnatural amino acid O-(2-bromoethyl)-tyrosine was incorporated into collagen by two-plasmid expression system. The results showed that high-purity collagen incorporated with unnatural amino acid could be obtained by induction with final concentration of 0.5 mmol/L IPTG and 0.06% arabinose at 25 °C for 24 hours. The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with unnatural amino acid and collagen molecule with cysteine mutation in pH 9.0 NH4HCO3 buffer, which formed aggregates with the largest molecular size up to 1 micrometre. The results pave the way for the design of recombinant collagen biomaterials.

Development of mammalian cell logic gates controlled by unnatural amino acids

SummaryMammalian cell logic gates hold great potential for wide-ranging applications. However, most of those currently available are controlled by drug(-like) molecules with inherent biological activities. To construct truly orthogonal circuits and artificial regulatory pathways, biologically inert molecules are ideal molecular switches. Here, we applied genetic code expansion and engineered logic gates controlled by two biologically inert unnatural amino acids. Genetic code expansion relies on orthogonal aminoacyl-tRNA synthetase/tRNA pairs for co-translational and site-specific unnatural amino acid incorporation conventionally in response to an amber (UAG) codon. By screening 11 quadruplet-decoding pyrrolysyl tRNA variants from the literature, we found that all variants decoding CUAG or AGGA tested here are functional in mammalian cells. Using a quadruplet-decoding orthogonal pair together with an amber-decoding pair, we constructed logic gates that can be successfully controlled by two different unnatural amino acids, expanding the scope of genetic code expansion and mammalian cell logic circuits.