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The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-l-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels–Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins., The membrane transporter BtuB is site-directedly spin labelled on the surface of living bacteria via Diels–Alder click chemistry.

Tyrosine sulfation, a post-translational modification that enhances extracellular protein-protein binding, plays a crucial role in various bacterial and viral infections. However, studying tyrosine-sulfated proteins can be difficult, because tyrosine sulfation of proteins often leads to mixtures of different sulfation patterns. In addition, efforts to engineer tyrosine-sulfated proteins is limited, because sulfation of the correct tyrosine is coupled to its local amino acid sequence contexts, termed sulfation motifs, which cannot be mutated without changing the sulfation efficiency. These complications can be resolved using an expanded genetic code system. A cell with an expanded genetic code for sulfotyrosine (sY) decouples sulfation motifs from the expression of homogenously sulfated proteins; this is accomplished through the site-specific incorporation of sY as a 21st amino acid at amber stop codons. Here, we use an upgrade expanded genetic code system to express high amounts of tyrosine-sulfated proteins for several applications. First, we expressed homogenously sulfated, bacterial RaxX peptide in order to validate its role in triggering immune response in rice plants. Second, we expressed all 32 possible sulfoforms of E51, one of the best sulfated anti-HIV antibodies at neutralizing HIV infections, and characterized their ability to bind to HIV's coat protein, gp120. We found several E51 sulfoforms that bind to gp120 with similar affinities as naturally sulfated E51 and determined key E51 residues for binding gp120. Lastly, we conducted multiple directed evolution experiments to improve the gp120-binding of E51 by repurposing E51's sulfation motifs with residues that bind to gp120.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Ne-(o-azidobenzyloxycarbonyl)- l -lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Protein phosphatases play an essential role in cell signaling; however, they remain understudied compared with protein kinases, in part due to a lack of appropriate tools. In order to provide conditional control over phosphatase function, we developed two different approaches for rendering MKP3 (a dual-specific phosphatase, also termed DUSP6) activatable by light. Specifically, we expressed the protein with strategically placed light-removable protecting groups in cells with an expanded genetic code. This allowed for the acute perturbation of the Ras/MAPK signaling pathway upon photoactivation in live cells. In doing so, we confirmed that MKP3 does not act as a thresholding gate for growth factor stimulation of the extracellular signal-regulated kinase (ESRK) pathway.

Nature, using proteins composed of approximately 20 amino acids repertoire, is able to perform a large number of admirable things, many of which we are still not quite able to mimic. However, it has to be said, that the set of chemical scaffolds available to the nature is somewhat limited. Therefore, it seems plausible to assume, that we could make things better simply by introducing novel scaffolds and exploring chemical space so far unavailable. It is exactly this idea upon which expanded genetic code is based on. To set ourselves free from limitations imposed by standard genetic code. This thesis will aim to provide an overview of various attempts to this problem and will try to do so in a distinct context of studying protein interactions. I will thus try to highlight methods by which we are currently able to expand genetic code, specific chemistries and physical properties of non-canonical amino acids and biophysical methods which can profit from genetic code expansion. Keywords: expanded genetic code, non-canonical amino acid, amber codon, fluores- cence, magnetic resonance, crosslinking, click chemistry

Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet non-trivial task for many research, such as when using Föster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case basis. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but it’s technically complicated and not suitable for some applications. Here we present a streamlined method that could enable dual site-specific protein labeling by using a tryptophan auxotroph of Escherichia coli to incorporate a naturally found tryptophan analog, 5-hydroxytryptophan into a recombinant protein. As a demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and site-specifically attached two different fluorophores, one on 5-hydroxytryptophan and another on a cysteine residue. This method is simple, generally applicable, efficient, and can serve as an alternative way for researchers who want to install an additional labeling site in their proteins.

In this comprehensive review, I focus on the twenty E. coli aminoacyl-tRNA synthetases and their ability to charge non-canonical amino acids (ncAAs) onto tRNAs. The promiscuity of these enzymes has been harnessed for diverse applications including understanding and engineering of protein function, creation of organisms with an expanded genetic code, and the synthesis of diverse peptide libraries for drug discovery. The review catalogues the structures of all known ncAA substrates for each of the 20 E. coli aminoacyl-tRNA synthetases, including ncAA substrates for engineered versions of these enzymes. Drawing from the structures in the list, I highlight trends and novel opportunities for further exploitation of these ncAAs in the engineering of protein function, synthetic biology, and in drug discovery.

Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA–protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA‐binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA–protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA‐binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA‐binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine‐based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA–protein interactions., UV irradiation is widely used in molecular biology to covalently crosslink RNA with RNA‐binding proteins; however, the yield of this process is usually low. We show that site‐selective labeling of an RNA‐binding protein with a diazirine‐based unnatural amino acid gives higher yields of the photo‐crosslinking product under milder irradiation conditions.

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.

Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure–activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N–H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.

SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins is valuable for both scientific and biomedical applications. To expand the structural diversity of available ncAAs and to reduce the burden of chemically synthesizing them, we have developed a general and simple biosynthetic method for genetically encoding novel ncAAs into recombinant proteins by feeding cells with economical commercially available or synthetically accessible aromatic thiols. We demonstrate that nearly 50 ncAAs with a diverse array of structures can be biosynthesized from these simple small-molecule precursors by hijacking the cysteine biosynthetic enzymes, and the resulting ncAAs can subsequently be incorporated into proteins via an expanded genetic code. Moreover, we demonstrate that bioorthogonal reactive groups such as aromatic azides and aromatic ketones can be incorporated into green fluorescent protein or a therapeutic antibody with high yields, allowing for subsequent chemical conjugation.

Links between trimethylamine-N-oxide (TMAO) and cardiovascular disease (CVD) have focused attention on mechanisms by which animal-based diets have negative health consequences. In a meta-analysis of data from foundational gut microbiome studies, we found evidence that specialized bacteria have and express a metabolic pathway that circumvents TMAO production and is often misannotated because it relies on genetic code expansion. This naturally occurring mechanism for TMAO attenuation is negatively correlated with CVD. Ultimately, these findings point to new avenues of research that could increase microbiome-informed understanding of human health and hint at potential biomedical applications in which specialized bacteria are used to curtail CVD development., Cardiovascular disease (CVD) has been linked to animal-based diets, which are a major source of trimethylamine (TMA), a precursor of the proatherogenic compound trimethylamine-N-oxide (TMAO). Human gut bacteria in the genus Bilophila have genomic signatures for genetic code expansion that could enable them to metabolize both TMA and its precursors without production of TMAO. We uncovered evidence that the Bilophila demethylation pathway is actively transcribed in gut microbiomes and that animal-based diets cause Bilophila to rapidly increase in abundance. CVD occurrence and Bilophila abundance in humans were significantly negatively correlated. These data lead us to propose that Bilophila, which is commonly regarded as a pathobiont, may play a role in mitigating cardiovascular disease. Human gut microbiomes have been shown to affect the development of a myriad of disease states, but mechanistic connections between diet, health, and microbiota have been challenging to establish. The hypothesis that Bilophila reduces cardiovascular disease by circumventing TMAO production offers a clearly defined mechanism with a potential human health impact, but investigations of Bilophila cell biology and ecology will be needed to fully evaluate these ideas. IMPORTANCE Links between trimethylamine-N-oxide (TMAO) and cardiovascular disease (CVD) have focused attention on mechanisms by which animal-based diets have negative health consequences. In a meta-analysis of data from foundational gut microbiome studies, we found evidence that specialized bacteria have and express a metabolic pathway that circumvents TMAO production and is often misannotated because it relies on genetic code expansion. This naturally occurring mechanism for TMAO attenuation is negatively correlated with CVD. Ultimately, these findings point to new avenues of research that could increase microbiome-informed understanding of human health and hint at potential biomedical applications in which specialized bacteria are used to curtail CVD development.

With the advances in the field of expanded genetic code, the application of non-canonical amino acid (ncAA) is considered an effective strategy for protein engineering. However, cumbersome and complicated selection schemes limit the extensive application of this technology in Saccharomyces cerevisiae. To address this issue, a simplified selection scheme with confident results was developed and tested in this study. Based on a mutation library derived from Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS), a logic gate in synthetic biology was used to optimize screening procedures. We found that an "and" gate was more suitable than an "or" gate for isolating aminoacyl-tRNA synthetase from S. cerevisiae. The successful incorporation of O-methyltyrosine (OMeY) proved the utility and efficiency of this new selection scheme. After a round of positive selection, several new OMeY-tRNA synthetase (OMeYRS) mutants were screened, and their incorporation efficiency was improved. Furthermore, we characterized the insertion of several tyrosine analogs into Herceptine Fab and discovered that OMeYRS and its mutants were polyspecific. One of these mutants showed an optimal performance to incorporate different ncAAs into recombinant proteins in S. cerevisiae; this mutant was cloned and transfected into mammalian cells, and the results proved its functionality in HEK293 cells. This study could expand the application of ncAA in S. cerevisiae to construct efficient yeast cell factories for producing natural and synthetic products.

By providing long-term protection against infectious diseases, vaccinations have significantly reduced death and morbidity worldwide. In the 21st century, (bio)technological advances have paved the way for developing prophylactic vaccines that are safer and more effective as well as enabling the use of vaccines as therapeutics to treat human diseases. Here, we provide a focused review of the utility of genetic code expansion as an emerging tool for the development of vaccines. Specifically, we discuss how the incorporation of immunogenic noncanonical amino acids can aid in eliciting immune responses against adverse self-proteins and highlight the potential of an expanded genetic code for the construction of replication-incompetent viruses. We close the review by discussing the future prospects and remaining challenges for the application of these approaches in the development of both prophylactic and therapeutic vaccines in the near future.

Genetic code expansion is one of the most powerful technologies in protein engineering. In addition to the 20 canonical amino acids, the expanded genetic code is supplemented by unnatural amino acids, which have artificial side chains that can be introduced into target proteins in vitro and in vivo. A wide range of chemical groups have been incorporated co‐translationally into proteins in single cells and multicellular organisms by using genetic code expansion. Incorporated unnatural amino acids have been used for novel structure‐function relationship studies, bioorthogonal labelling of proteins in cellulo for microscopy and in vivo for tissue‐specific proteomics, the introduction of post‐translational modifications and optical control of protein function, to name a few examples. In this Minireview, the development of genetic code expansion technology is briefly introduced, then its applications in neurobiology are discussed, with a focus on studies using mammalian cells and mice as model organisms., Do what comes unnaturally: The genetic code has been expanded to include unnatural amino acids (UAAs). Thanks to their unique side chains, UAAs confer new properties on target proteins. These allow us to modify biological processes with unprecedented molecular precision. The applications of this technology in neurobiology are the focus of this Minireview.

With few exceptions, natural proteins are built from only 20 canonical (proteogenic) amino acids which limits the functionality and accordingly the properties they can possess. Genetic code expansion, i.e. the creation of codons and the machinery needed to assign them to non-canonical amino acids (ncAAs), promises to enable the discovery of proteins with novel properties that are otherwise difficult or impossible to obtain. One approach to expanding the genetic code is to expand the genetic alphabet via the development of unnatural nucleotides that pair to form an unnatural base pair (UBP). Semi-synthetic organisms (SSOs) - organisms that stably maintain the UBP, transcribe its component nucleotides into RNA, and use it to translate proteins, - would have available to them new codons and the anticodons needed to assign them to ncAAs. This review summarizes the development of a family of UBPs, their use to create SSOs, and the optimization and application of the SSOs to produce candidate therapeutic proteins with improved properties that are now undergoing evaluation in clinical trials.

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine O-succinyltransferase ( metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with ( p-benzoylphenyl)alanine (pBzF), increases the melting temperature of E. coli metA by 21 °C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.

Current methods to expand the genetic code enable site-specific incorporation of non-canonical amino acids (ncAAs) into proteins in eukaryotic and prokaryotic cells. However, current methods are limited by the number of codons possible, their orthogonality, and possibly their effects on protein synthesis and folding. An alternative approach relies on unnatural base pairs to create a virtually unlimited number of genuinely new codons that are efficiently translated and highly orthogonal because they direct ncAA incorporation using forces other than the complementary hydrogen bonds employed by their natural counterparts. This review outlines progress and achievements made towards developing a functional unnatural base pair and its use to generate semi-synthetic organisms with an expanded genetic alphabet that serves as the basis of an expanded genetic code.

In nature, biological processes are regulated with precise spatial and temporal resolution at the molecular, cellular, and organismal levels. In order to perturb and manipulate these processes, optically controlled chemical tools have been developed and applied in living systems. The use of light as an external trigger provides spatial and temporal control with minimal adverse effects. Incorporation of light-responsive amino acids into proteins in cells and organisms with an expanded genetic code has enabled the precise activation/deactivation of numerous, diverse proteins, such as kinases, nucleases, proteases, and polymerases. Using unnatural amino acids to generate light-triggered proteins enables a rational engineering approach that is based on mechanistic and/or structural information. This review focuses on the most recent developments in the field, including technological advances and biological applications.

Synthetic biology has been revolutionizing the biopharmaceutical industry from drug discovery, clinical development to the manufacturing of biopharmaceuticals. As one of its most promising areas, genetic incorporation of noncanonical amino acids (ncAA) into proteins via an expanded genetic code emerged with great promise in the pharmaceutical industry recently with multiple therapeutic candidates tested in human clinical trials and one approved veterinary drug. Expanded building blocks enable proteins to have new or modified functions, providing ample opportunities for innovative or improved medicines. Here we review the current progress in the technology platforms for manufacturing ncAA-containing therapeutics and the pharmaceutical applications of an expanded genetic code.

In nature, 64 different codons encode 20 canonical amino acids into proteins with few exemptions. Genetic code expansion technique allows site-specific incorporation of diverse noncanonical amino acids (ncAAs) through nonsense codons suppression into recombinant proteins or the proteomes of living cells. To date, hundreds of ncAAs have been successfully incorporated into a variety of proteins in different species, and their applications have recently expanded from fundamental research to biotherapeutics. Here, we reviewed and discussed the recent progress in the therapeutic applications of an expanded genetic code. On one hand, genetic incorporations of ncAAs with bio-orthogonal reactive groups allow site-specific modifications of protein therapeutics and cell therapeutics with unprecedented quality control, safety and efficacy. On the other hand, the nonsense codon readthrough ability makes it possible for synthetic biologists to exploit new generation biotherapeutics.

Selenoproteins containing the 21(st) amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low pKa, high reactivity and redox property of Sec also afford unique routes to protein modification and engineering. However, natural Sec incorporation requires idiosyncratic translational machineries that are dedicated to Sec and species-dependent, which makes it challenging to recombinantly prepare selenoproteins with high Sec specificity. As a consequence, the function of half of human selenoproteins remain unclear, and Sec-based protein manipulation has been greatly hampered. Here we report a new general method enabling the site-specific incorporation of Sec into proteins in E. coli. An orthogonal tRNA(Pyl)-ASecRS was evolved to specifically incorporate Se-allyl selenocysteine (ASec) in response to the amber codon, and the incorporated ASec was converted to Sec in high efficiency through palladium-mediated cleavage under mild conditions compatible with proteins and cells. This approach completely obviates the natural Sec-dedicated factors, thus allowing various selenoproteins, regardless of Sec position and species source, to be prepared with high Sec specificity and enzyme activity, as shown by the preparation of human thioredoxin and glutathione peroxidase 1. Sec-selective labeling in the presence of Cys was also demonstrated on the surface of live E. coli cells. The tRNA(Pyl)-ASecRS pair was further used in mammalian cells to incorporate ASec, which was converted into Sec by palladium catalyst in cellulo. This robust and versatile method should greatly facilitate the study of diverse natural selenoproteins and the engineering of proteins in general via site-specific introduction of Sec.

Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded Escherichia coli strains with improved fitness that are superior hosts for recombinant selenoprotein production. We apply an engineered β-lactamase containing an essential diselenide bond to enforce selenocysteine dependence during continuous evolution of recoded E. coli strains. Evolved strains maintain an expanded genetic code indefinitely. We engineer a fluorescent reporter to quantify selenocysteine incorporation in vivo and show complete decoding of UAG codons as selenocysteine. Replacement of native, labile disulfide bonds in antibody fragments with diselenide bonds vastly improves resistance to reducing conditions. Highly seleno-competent bacterial strains enable industrial-scale selenoprotein expression and unique diselenide architecture, advancing our ability to customize the selenoproteome.

Cell-lineage tracing is used to study embryo development and stem-cell differentiation as well as to document tumor cell heterogeneity. Cre recombinase-mediated cell labeling is the preferred approach; however, its utility is restricted by when and where DNA recombination takes place. We generated a photoactivatable Cre recombinase by replacing a critical residue in its active site with a photocaged lysine derivative through genetic code expansion in zebrafish embryos. This allows high spatiotemporal control of DNA recombination by using 405 nm irradiation. Importantly, no background activity is seen before irradiation, and, after light-triggered removal of the caging group, Cre recombinase activity is restored. We demonstrate the utility of this tool as a cell-lineage tracer through its activation in different regions and at different time points in the early embryo. Direct control of Cre recombinase by light will allow more precise DNA recombination, thereby enabling more nuanced studies of metazoan development and disease.

Tyrosine sulfation is a crucial post-translational modification for certain antibodies that neutralize HIV. One of the most neutralizing sulfated anti-HIV antibodies, E51, contains a region in its VHCDR3 loop with five tyrosine (Tyr) residues, which are hypothesized to be partially or fully sulfated to bind to HIV’s gp120 coat protein. However, the gp120-binding contribution of each sulfate or more complex sulfation patterns is unknown. In addition, natural sulfation of Tyr-rich loops usually yields a mixture of multiply sulfated products, complicating attempts to dissect the function of individual E51 sulfoforms with unique sulfation patterns. Here, we use an upgraded expanded genetic code for sulfotyrosine (sY) to express homogeneous E51 sulfoforms containing up to five sulfates. Through characterization of the 32 possible sulfoforms of E51, we show that only a subset of E51 sulfoforms with two, three, or four sYs bind to gp120 with potency similar to that of post-translationally sulfated E51, which we ...

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of Methanocaldococcus jannaschii (Mj) tRNATyr-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native E. coli RNA-modifying ...

Background To find experimental validation for electrostatic interactions essential for catalytic reactions represents a challenge due to practical limitations in assessing electric fields within protein structures. Scope of review This review examines the applications of non-canonical amino acids (ncAAs) as genetically encoded probes for studying the role of electrostatic interactions in enzyme catalysis. Major conclusions ncAAs constitute sensitive spectroscopic probes to detect local electric fields by exploiting the vibrational Stark effect (VSE) and thus have the potential to map the protein electrostatics. General significance Mapping the electrostatics in proteins will improve our understanding of natural catalytic processes and, in beyond, will be helpful for biocatalyst engineering. This article is part of a Special Issue entitled “Biochemistry of Synthetic Biology - Recent Developments” Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.