Incorporation Of Unnatural Amino Acids Research Articles

Biomolecular simulation based machine learning models accurately predict sites of tolerability to the unnatural amino acid acridonylalanine

The incorporation of unnatural amino acids (Uaas) has provided an avenue for novel chemistries to be explored in biological systems. However, the successful application of Uaas is often hampered by site-specific impacts on protein yield and solubility. Although previous efforts to identify features which accurately capture these site-specific effects have been unsuccessful, we have developed a set of novel Rosetta Custom Score Functions and alternative Empirical Score Functions that accurately predict the effects of acridon-2-yl-alanine (Acd) incorporation on protein yield and solubility. Acd-containing mutants were simulated in PyRosetta, and machine learning (ML) was performed using either the decomposed values of the Rosetta energy function, or changes in residue contacts and bioinformatics. Using these feature sets, which represent Rosetta score function specific and bioinformatics-derived terms, ML models were trained to predict highly abstract experimental parameters such as mutant protein yield and solubility and displayed robust performance on well-balanced holdouts. Model feature importance analyses demonstrated that terms corresponding to hydrophobic interactions, desolvation, and amino acid angle preferences played a pivotal role in predicting tolerance of mutation to Acd. Overall, this work provides evidence that the application of ML to features extracted from simulated structural models allow for the accurate prediction of diverse and abstract biological phenomena, beyond the predictivity of traditional modeling and simulation approaches.

Breaker peptides against amyloid-β aggregation: a potential therapeutic strategy for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, for whichblocking the early steps of extracellular misfolded amyloid-β (Aβ) aggregation is a promising therapeutic approach. However, the pathological features of AD progression includethe accumulation of intracellular tau protein, membrane-catalyzed cell deathand the abnormal deposition of Aβ. Here, we focus on anti-amyloid breaker peptides derived from the Aβ sequence and non-Aβ-based peptidescontaining both natural and modified amino acids. Critical aspects of the breaker peptides include N-methylation, conformational restriction through cyclization, incorporation of unnatural amino acid, fluorinated molecules, polymeric nanoparticles andPEGylation. This review confers a general idea of such breaker peptides with in vitro and in vivo studies, which may advance our understanding of AD pathology and develop an effective treatment strategy against AD.

Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids.

Approximately 11% of monogenic diseases involve nonsense mutations that are caused by premature termination codons. These codons can in principle be read-through via the site-specific incorporation of unnatural amino acids to generate full-length proteins with minimal loss of function. Here we report that aminoacyl-tRNA-synthase-tRNA pairs specific for the desired unnatural amino acids can be used to read through a nonsense mutation in the dystrophin gene. We show partial restoration of dystrophin expression in differentiated primary myoblasts (from a mdx mouse model and a patient with Duchenne muscular dystrophy), and restoration of muscle function in two mouse models: mdx mice, via viral delivery of the engineered tRNA-synthase-tRNA pair intraperitoneally or intramuscularly and of the associated unnatural amino acid intraperitoneally; and mice produced by crossing mdx mice and transgenic mice with a chromosomally integrated pair, via intraperitoneal delivery of the unnatural amino acid. The incorporation of unnatural amino acids to restore endogenous protein expression could be explored for therapeutic use.

A linear DNA template-based framework for site-specific unnatural amino acid incorporation

Site-specific incorporation of unnatural amino acids (UNAAs) into proteins using an orthogonal translation system (OTS) has expanded the scope of protein-coding chemistry. The key factor affecting UNAA embedding efficiency is the orthogonality of the OTS. Compared to traditional cell systems, cell-free systems are more convenient to control the reaction process and improve the utilization rate of UNAA. In this study, a linear DNA template-based cell-free unnatural protein synthesis system for rapid high-throughput screening and evolution was proposed. A total of 14 cell extracts were selected for screening out cell extract with high expression level. The result showed that EcAR7 ΔA ΔSer cell extract was optimal for the cell-free system. In addition, the screening results of four UNAAs, p-propargyloxy-l-phenylalanine (pPaF), p-azyl-phenylalanine (pAzF), p-acetyl-l-phenylalanine (pAcF), and p-benzoyl-l-phenylalanine (pBpF), showed that o-aaRS and o-tRNA of pPaF had good orthogonality. A new pair of corresponding o-aaRS and o-tRNA for pBpF was screened out. These results proved that this method could speed up the screening of optimal OTS components for UNAAs with versatile functions.

Characterization of Fluorescent Proteins with Intramolecular Photostabilization*.

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count‐rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet‐state quenchers or redox‐active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α‐GFP for attachment of photostabilizer‐maleimides on the β‐barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α‐GFP‐photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet‐energy of azobenzene might be crucial for triplet‐quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation.

To View Your Biomolecule, Click inside the Cell.

The surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades. Involving chemical partners that specifically react together in highly complex biological fluids, this branch of chemistry now allows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies. Chemical reporter strategies include metabolic glycan labelling, site-specific incorporation of unnatural amino acids in proteins, and post-synthetic labelling of nucleic acids. While a majority of literature reports mark cell-surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes a more challenging task. Owing to limiting factors such as membrane permeability of reagents, fluorescence background due to hydrophobic interactions and off-target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes, these strategies need mindful planning to achieve success. In this review, we discuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets.

To View Your Biomolecule, Click inside the Cell

AbstractThe surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades. Involving chemical partners that specifically react together in highly complex biological fluids, this branch of chemistry now allows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies. Chemical reporter strategies include metabolic glycan labelling, site‐specific incorporation of unnatural amino acids in proteins, and post‐synthetic labelling of nucleic acids. While a majority of literature reports mark cell‐surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes a more challenging task. Owing to limiting factors such as membrane permeability of reagents, fluorescence background due to hydrophobic interactions and off‐target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes, these strategies need mindful planning to achieve success. In this review, we discuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets.

Genetic Code Expansion: A Brief History and Perspective.

Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.

In Situ Assay of Proteins Incorporated with Unnatural Amino Acids in Single Living Cells by Differenced Resonance Light Scattering Correlation Spectroscopy.

Site-specific incorporation of unnatural amino acids (UAAs) into target proteins (UAA-proteins) provides the unprecedented opportunities to study cell biology and biomedicine. However, it is a big challenge to in situ quantitatively determine the expression level of UAA-proteins due to serious interferences from autofluorescence, background scattering, and different viscosity in living cells. Here, we proposed a novel single nanoparticle spectroscopy method, differenced resonance light scattering correlation spectroscopy (D-RLSCS), to measure the UAA-proteins in single living cells. The D-RLSCS principle is based on the simultaneous measurement of the resonance scattering light fluctuation of a single gold nanoparticle (GNP) in two detection channels irradiated by two coaxial laser beams and then autocorrelation analysis on the differenced fluctuation signals between two channels. D-RLSCS can avoid the interferences from intracellular background scattering and provide the concentration and rotational and translational diffusion information of GNPs in solution or in living cells. Furthermore, we proposed a parameter, the ratiometric diffusion time and found that this parameter is proportional to the square of particle size. The theoretical and experimental results demonstrated that the ratiometric diffusion time was not influenced by the intracellular viscosity. This method was successfully applied for in situ quantification of the UAA-protein within single living cells based on the increase in the ratiometric diffusion time of nanoprobes bound with proteins. Using UAA-EGFP (enhanced green fluorescent protein) as a model, we observed the significant difference in the UAA-protein concentrations at different positions in single living cells.

Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid.

To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.

Assessing Local Environments in Green Fluorescent Protein using Unnatural Amino Acid 4‐Cyano‐L‐Phenylalanine

An increasingly useful method of studying local electrostatic environments within protein molecules is through the incorporation of unnatural amino acids (UAAs) containing distinguishable chemical bonds with specific features in the infrared spectrum (IR) that change based on local environments. Of the existing UAAs, 4-cyano-L-phenylalanine (pCNF) is arguably one of the most useful vibrational reporters due to the fact that it is easily and affordably synthesized, it is well-studied, and its nitrile stretch appears in a biologically quiet region of the IR spectrum. In this study, pCNF has been genetically incorporated individually into three distinct sites of superfolder green fluorescent protein (sfGFP) via the amber codon suppression methodology. According to the wild type crystal structure of sfGFP, each of the selected sites are expected to have a unique solvation environment. Temperature-dependent infrared (IR) spectroscopy was utilized in order to test assess the local solvation environments of each of these sites. By measuring the nitrile stretching frequency as a function of temperature, the degree to which the incorporated pCNF participates in hydrogen bonding was determined. IR data and X-ray crystal structures from these constructs will be presented.

Current Advances in Covalent Stabilization of Macromolecular Complexes for Structural Biology.

Structural characterization of macromolecular assemblies is often limited by the transient nature of the interactions. The development of specific chemical tools to covalently tether interacting proteins to each other has played a major role in various fundamental discoveries in recent years. To this end, protein engineering techniques such as mutagenesis, incorporation of unnatural amino acids, and methods using synthetic substrate/cosubstrate derivatives were employed. In this review, we give an overview of both commonly used and recently developed biochemical methodologies for covalent stabilization of macromolecular complexes enabling structural investigation via crystallography, nuclear magnetic resonance, and cryo-electron microscopy. We divided the strategies into nonenzymatic- and enzymatic-driven cross-linking and further categorized them in either naturally occurring or engineered covalent linkage. This review offers a compilation of recent advances in diverse scientific fields where the structural characterization of macromolecular complexes was achieved by the aid of intermolecular covalent linkage.

Amber codon is genetically unstable in generation of premature termination codon (PTC)-harbouring Foot-and-mouth disease virus (FMDV) via genetic code expansion

ABSTRACT The foot-and-mouth disease virus (FMDV) is the causative agent of FMD, a highly infectious and devastating viral disease of domestic and wild cloven-hoofed animals. FMD affects livestock and animal products’ national and international trade, causing severe economic losses and social consequences. Currently, inactivated vaccines play a vital role in FMD control, but they have several limitations. The genetic code expansion technology provides powerful strategies for generating premature termination codon (PTC)-harbouring virus as a live but replication-incompetent viral vaccine. However, this technology has not been explored for the design and development of new FMD vaccines. In this study, we first expanded the genetic code of the FMDV genome via a transgenic cell line containing an orthogonal translation machinery. We demonstrated that the transgenic cells stably integrated the orthogonal pyltRNA/pylRS pair into the genome and enabled efficient, homogeneous incorporation of unnatural amino acids into target proteins in mammalian cells. Next, we constructed 129 single-PTC FMDV mutants and four dual-PTC FMDV mutants after considering the tolerance, location, and potential functions of those mutated sites. Amber stop codons individually substituted the selected amino acid codons in four viral proteins (3D, L, VP1, and VP4) of FMDV. We successfully rescued PTC-FMDV mutants, but the amber codon unexpectedly showed a highly degree of mutation rate during PTC-FMDV packaging and replication. Our findings highlight that the genetic code expansion technology for the generation of PTC-FMD vaccines needs to be further improved and that the genetic stability of amber codons during the packaging and replication of FMDV is a concern.

The Benefits of Unnatural Amino Acid Incorporation as Protein Labels for Single Molecule Localization Microscopy.

Single Molecule Localization Microscopy (SMLM) is an imaging method that allows for the visualization of structures smaller than the diffraction limit of light (~200 nm). This is achieved through techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM). A large part of obtaining ideal imaging of single molecules is the choice of the right fluorescent label. An upcoming field of protein labeling is incorporating unnatural amino acids (UAAs) with an attached fluorescent dye for precise localization and visualization of individual molecules. For this technique, fluorescent probes are conjugated to UAAs and are introduced into the protein of interest (POI) as a label. Here we contrast this labeling method with other commonly used protein-based labeling methods such as fluorescent proteins (FPs) or self-labeling tags such as Halotag, SNAP-tags, and CLIP-tags, and highlight the benefits and shortcomings of the site-specific incorporation of UAAs coupled with fluorescent dyes in SMLM.

Incorporation of Unnatural Amino Acid into Antibody Fragment for Creating a Stable Antibody Drug Conjugate

The traditional chemotherapy drug has been used as a standard cancer treatment, however it has resulted a modest survival benefit and damaged non-cancerous cells. Thus, the novel strategies which can improve selectivity and specificity in chemotherapy are urgently needed. Antibody drug conjugate (ADC) combines monoclonal antibody and cytotoxic drug is a potential regimen as targeted therapy. However, the heterogeneous mixtures has been observed using the current ADC methods. Here, we develop the strategy for generation a stable ADC utilising modified single chain antibody fragment (scFv) containing azide group for click chemistry reaction with alkyne containing cytotoxic drug. This research focused on targeting prostate cancer as a model disease utilising targeting prostate specific membrane antigen (PSMA) receptor which is overexpressed in all prostate cancer stages. The unnatural amino acid para-azido phenyl alanine (pAzF) has been successfully incorporated into anti-PSMA J591 scFv and specifically bound and internalised into PSMA positive cancer cells. This mutant scFv were also successfully conjugated into a linker containing cyclo-alkyne, DBCO-PEG4-DBCO as a model for creating ADC through copper-free click chemistry reaction. This bioconjugation method is promising as a versatile strategy for generating a stable ADC to improve therapeutic efficacy in cancer treatment.

Genetic Code Expansion of Vibrio natriegens.

Escherichia coli has been considered as the most used model bacteria in the majority of studies for several decades. However, a new, faster chassis for synthetic biology is emerging in the form of the fast-growing gram-negative bacterium Vibrio natriegens. Different methodologies, well established in E. coli, are currently being adapted for V. natriegens in the hope to enable a much faster platform for general molecular biology studies. Amongst the vast technologies available for E. coli, genetic code expansion, the incorporation of unnatural amino acids into proteins, serves as a robust tool for protein engineering and biorthogonal modifications. Here we designed and adapted the genetic code expansion methodology for V. natriegens and demonstrate an unnatural amino acid incorporation into a protein for the first time in this organism.

Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria.

Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment - typically at a single site or at a few sites per protein - of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l-phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production - using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain - of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.

The Role Trna-Mediated Regulation of Ribosome Dynamics Plays in Frameshifting by +1 Frameshift-Inducing Trnas

The ability to incorporate unnatural amino acids (UAAs) into proteins promises novel tools for probing protein structure and function. +1 frameshift-inducing (+1FI) tRNAs, which have evolved to promote +1 ribosomal frameshifting in order to correct errors at quadruplet codons resulting from insertion or deletion mutations, have recently emerged as attractive tools for encoding UAAs at engineered quadruplet codons. Despite decades of study, however, the molecular mechanisms through which +1FI tRNAs promote +1 frameshifting remain elusive, impeding efforts to optimize the cellular translation machinery so as to increase the efficiency of UAA incorporation.

Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis.

The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the C=O group through C–C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provide a powerful method for adding valuable reactivity and expanding possible function in proteins. Here, following a survey of methods for HCF2· generation, we show that reductive photoredox catalysis enables mild radical-mediated difluoromethylation-hydrolysis of native protein residues as an effective method for carbonylation. Inherent selectivity of HCF2· allowed preferential modification of Trp residues. The resulting C-2-difluoromethylated Trp undergoes Reimer-Tiemann-type dehalogenation providing highly effective spontaneous hydrolytic collapse in proteins to carbonylated HC(O)-Trp (C-formyl-Trp = CfW) residues. This new, unnatural protein residue CfW not only was found to be effective in bioconjugation, ligation, and labeling reactions but also displayed strong “red-shifting” of its absorption and fluorescent emission maxima, allowing direct use of Trp sites as UV–visualized fluorophores in proteins and even cells. In this way, this method for the effective generation of masked formyl-radical “HC(O)·” equivalents enables first examples of C–C bond-forming carbonylation in proteins, thereby expanding the chemical reactivity and spectroscopic function that may be selectively and post-translationally “edited” into biology.

The Ras dimer structure.

Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein–protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target.