Diversity and modularity of tyrosine-accepting tRNA-like structures.

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLSTyr) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5' domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLSTyr compared to those in Bromoviridae Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLSTyr and the 3' domain from a hordeivirus TLSTyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLSTyrs performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLSTyr Finally, these structurally and phylogenetically divergent types of TLSTyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures.

Orthogonal translation with 5-cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding.

Orthogonal translation is an efficient tool that provides many valuable spectral probes capable of covering different parts of the electromagnetic spectrum and thus enabling parameterization of various structural and dynamic phenomena in proteins. In this context, nitrile-containing tryptophan analogs are very useful probes to study local electrostatics and hydrogen bonding in both rigid and dynamic environments. Here, we report a semi-rational approach to engineer a tyrosyl-tRNA synthetase (TyrRS) variant of Methanocaldococcus jannaschii capable of incorporating 5-cyanotryptophan (5CNW) via orthogonal translation. We combined one round of the well-established positive selection system with saturation mutagenesis at preselected TyrRS positions, resulting in a novel 5CNW-specific enzyme that also exhibits high substrate tolerance to other aromatic noncanonical amino acids. We demonstrated the utility of our orthogonal pair by inserting 5CNW into the cyanobacteriochrome Slr1393g3, a bilin-binding photosensor of the phytochrome superfamily. The nitrile (CN) group of the inserted 5CNW provides non-invasive labeling in the local structural context while yielding information on local electrostatics and hydrogen bonding by IR spectroscopy. 5CNW is a versatile probe that can be used for both static and dynamic measurements.

Nuclear ATR lysine-tyrosylation protects against heart failure by activating DNA damage response.

Dysregulated amino acid increases the risk for heart failure (HF) via unclear mechanisms. Here, we find that increased plasma tyrosine and phenylalanine levels are associated with HF. Increasing tyrosine or phenylalanine by high-tyrosine or high-phenylalanine chow feeding exacerbates HF phenotypes in transverse aortic constriction and isoproterenol infusion mice models. Knocking down phenylalanine dehydrogenase abolishes the effect of phenylalanine, indicating that phenylalanine functions by converting to tyrosine. Mechanistically, tyrosyl-tRNA synthetase (YARS) binds to ataxia telangiectasia and Rad3-related gene (ATR), catalyzes lysine tyrosylation (K-Tyr) of ATR, and activates the DNA damage response (DDR) in the nucleus. Increased tyrosine inhibits the nuclear localization of YARS, inhibits the ATR-mediated DDR, accumulates DNA damage, and elevates cardiomyocyte apoptosis. Enhancing ATR K-Tyr by overexpressing YARS, restricting tyrosine, or supplementing tyrosinol, a structural analog of tyrosine, promotes YARS nuclear localization and alleviates HF in mice. Our findings implicate facilitating YARS nuclear translocation as a potential preventive and/or interfering measure against HF.

Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography.

Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its specific variant of Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (oNBTyrRS), and then demonstrated its potential availability for time-resolved X-ray crystallography. The substrate-bound crystal structure of oNBTyrRS at a 2.79 Å resolution indicated that the replacement of tyrosine and leucine at positions 32 and 65 by glycine (Tyr32Gly and Leu65Gly, respectively) and Asp158Ser created sufficient space for entry of the bulky substitute into the amino acid binding pocket, while Glu in place of Leu162 formed a hydrogen bond with the nitro moiety of oNBTyr. We also produced an oNBTyr-containing lysozyme through a cell-free protein synthesis system derived from the Escherichia coli B95. ΔA strain with the UAG codon reassigned to the nonnatural amino acid. Another crystallographic study of the caged protein showed that the site-specifically incorporated oNBTyr was degraded to tyrosine by light irradiation of the crystals. Thus, cell-free protein synthesis of caged proteins with oNBTyr could facilitate time-resolved structural analysis of proteins, including medically important membrane proteins.

Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

A viral RNA hijacks host machinery using dynamic conformational changes of a tRNA-like structure.

Viruses require multifunctional structured RNAs to hijack their host's biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo­electron microscopy (cryo-EM), we visualized the structure of the mysterious viral transfer RNA (tRNA)­like structure (TLS) from the brome mosaic virus, which affects replication, translation, and genome encapsidation. Structures in isolation and those bound to tyrosyl-tRNA synthetase (TyrRS) show that this ~55-kilodalton purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs substantially from that of tRNA. Our study reveals how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions, and we highlight the utility of cryo-EM for visualizing small, conformationally dynamic structured RNAs.

The recurrent missense mutation p.(Arg367Trp) in YARS1 causes a distinct neurodevelopmental phenotype.

Pathogenic variants in aminoacyl-tRNA synthetases (ARS1) cause a diverse spectrum of autosomal recessive disorders. Tyrosyl tRNA synthetase (TyrRS) is encoded by YARS1 (cytosolic, OMIM*603,623) and is responsible of coupling tyrosine to its specific tRNA. Next to the enzymatic domain, TyrRS has two additional functional domains (N-Terminal TyrRSMini and C-terminal EMAP-II-like domain) which confer cytokine-like functions. Mutations in YARS1 have been associated with autosomal-dominant Charcot-Marie-Tooth (CMT) neuropathy type C and a heterogenous group of autosomal recessive, multisystem diseases. We identified 12 individuals from 6 families with the recurrent homozygous missense variant c.1099C > T;p.(Arg367Trp) (NM_003680.3) in YARS1. This variant causes a multisystem disorder with developmental delay, microcephaly, failure to thrive, short stature, muscular hypotonia, ataxia, brain anomalies, microcytic anemia, hepatomegaly, and hypothyroidism. In silico analyses show that the p.(Arg367Trp) does not affect the catalytic domain responsible of enzymatic coupling, but destabilizes the cytokine-like C-terminal domain. The phenotype associated with p.(Arg367Trp) is distinct from the other biallelic pathogenic variants that reside in different functional domains of TyrRS which all show some common, but also divergent clinical signs [(e.g., p.(Phe269Ser)-retinal anomalies, p.(Pro213Leu)/p.(Gly525Arg)-mild ID, p.(Pro167Thr)-high fatality)]. The diverse clinical spectrum of ARS1-associated disorders is related to mutations affecting the various non-canonical domains of ARS1, and impaired protein translation is likely not the exclusive disease-causing mechanism of YARS1- and ARS1-associated neurodevelopmental disorders. KEY MESSAGES: The missense variant p.(Arg367Trp) in YARS1 causes a distinct multisystem disorder. p.(Arg367Trp) affects a non-canonical domain with cytokine-like functions. Phenotypic heterogeneity associates with the different affected YARS1 domains. Impaired protein translation is likely not the exclusive mechanism of ARS1-associated disorders.

Interplay of substrate polymorphism and conformational plasticity of Plasmodium tyrosyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are an indispensable component of ribosomal protein translational machinery and Plasmodium Tyrosyl-tRNA synthetase (PfTyrRS) is a validated drug target. This manuscript illustrates the dynamic conformational landscape of PfTyrRS in the context of substrate binding. Molecular dynamics simulations of PfTyrRS in the presence and absence of ligand show conformational heterogeneity for both the protein and the bound ligand. Diverse conformations for the evolutionarily conserved ATP binding motif (KMSKS) have been observed in both apo- and holo PfTyrRS. Further, the presented attributes of the tyrosyl-adenylate conformational sub-states in situ along with their implications on the strength of intermolecular interactions would be a pertinent benchmark for molecular design studies. In addition, an analysis of the ligand hydration pattern foregrounds the structurally conserved water-mediated inter-molecular interactions. The quantitative assessment of the conformational landscape, based on the fluctuations of the distance between the ligand binding pockets, of apo-PfTyrRS and holo-PfTyrRS highlights the nature of diversity in conformational sampling for the two cases. Evidently, the holo-PfTyrRS adopts a rather compact conformation compared to the apo-PfTyrRS. An intriguing asymmetry in the dynamics of the two monomers is contextualized with the functional asymmetry of the symmetrically dimeric PfTyrRS. Importantly, the network of non-bonded contacts in the apo- and holo- simulated ensembles has been analyzed. The graph-theoretic analysis-based novel insights concerning the nature of information flow as a function of ligation state would prove valuable in understanding PfTyrRS functions. The results presented here contend that understanding allostery in PfTyrRS is essential to astutely design structure-based inhibitors.

Crystal structure of Nanoarchaeum equitans tyrosyl-tRNA synthetase and its aminoacylation activity toward tRNATyr with an extra guanosine residue at the 5'-terminus.

tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5'-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5'-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 Å resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5'-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.

Structural Robustness Affects the Engineerability of Aminoacyl-tRNA Synthetases for Genetic Code Expansion.

The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The Escherichia coli-derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs have been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the structural robustness of EcTyrRS being lower than that of MjTyrRS. We show that the thermostability of engineered TyrRS mutants is generally significantly lower than those of their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homologue from a thermophilic bacterium, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.

Tyrosine tRNA synthetase as a novel extracellular immunomodulatory protein in Streptococcus anginosus.

Streptococcus anginosus is frequently detected in patients with infective endocarditis, abscesses or oral cancer. Although S. anginosus is considered the causative pathogen of these diseases, the pathogenic mechanisms of the bacterium have remained unclear. Previously, we suggested that an extracellular antigen from S. anginosus (SAA) serves as a pathogenic factor by inducing nitric oxide production in murine macrophages. In the present study, we identified SAA using LC-MS/MS and assessed the biological activities of His-tagged recombinant SAA in murine macrophages. SAA was identified as a tyrosine tRNA synthetase (SaTyrRS) that was isolated from the extracellular fraction of S. anginosus but not from other oral streptococci. In addition, inducible nitric oxide synthase and TNF-α mRNA expression was induced in recombinant SaTyrRS-stimulated murine macrophages. However, their mRNA expression was not induced in macrophages stimulated with truncated or heat-inactivated recombinant SaTyrRS, and the activation motif was identified as Arg264-Thr270. Consequently, these results indicated that SaTyrRS could be a novel and specific immunomodulatory protein in S. anginosus.

Functional genetic encoding of sulfotyrosine in mammalian cells.

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Phenyltriazole-functionalized sulfamate inhibitors targeting tyrosyl- or isoleucyl-tRNA synthetase.

Antimicrobial resistance is considered as one of the major threats for the near future as the lack of effective treatments for various infections would cause more deaths than cancer by 2050. The development of new antibacterial drugs is considered as one of the cornerstones to tackle this problem. Aminoacyl-tRNA synthetases (aaRSs) are regarded as good targets to establish new therapies. Apart from being essential for cell viability, they are clinically validated. Indeed, mupirocin, an isoleucyl-tRNA synthetase (IleRS) inhibitor, is already commercially available as a topical treatment for MRSA infections. Unfortunately, resistance developed soon after its introduction on the market, hampering its clinical use. Therefore, there is an urgent need for new cellular targets or improved therapies. Follow-up research by Cubist Pharmaceuticals led to a series of selective and in vivo active aminoacyl-sulfamoyl aryltetrazole inhibitors targeting IleRS (e.g. CB 168). Here, we describe the synthesis of new IleRS and TyrRS inhibitors based on the Cubist Pharmaceuticals compounds, whereby the central ribose was substituted for a tetrahydropyran ring. Various linkers were evaluated connecting the six-membered ring with the base-mimicking part of the synthesized analogues. Out of eight novel molecules, a three-atom spacer to the phenyltriazole moiety, which was established using azide-alkyne click chemistry, appeared to be the optimized linker to inhibit IleRS. However, 11 (Ki,app = 88 ± 5.3 nM) and 36a (Ki,app = 114 ± 13.5 nM) did not reach the same level of inhibitory activity as for the known high-affinity natural adenylate-intermediate analogue isoleucyl-sulfamoyl adenosine (IleSA, CB 138; Ki,app = 1.9 ± 4.0 nM) and CB 168, which exhibit a comparable inhibitory activity as the native ligand. Therefore, 11 was docked into the active site of IleRS using a known crystal structure of T. thermophilus in complex with mupirocin. Here, we observed the loss of the crucial 3'- and 4'- hydroxyl group interactions with the target enzyme compared to CB 168 and mupirocin, which we suggest to be the reason for the limited decrease in enzyme affinity. Despite the lack of antibacterial activity, we believe that structurally optimizing these novel analogues via a structure-based approach could ultimately result in aaRS inhibitors which would help to tackle the antibiotic resistance problem.

Role of disordered regions in transferring tyrosine to its cognate tRNA.

Aminoacyl tRNA synthetase (AARS) plays an important role in transferring each amino acid to its cognate tRNA. Specifically, tyrosyl tRNA synthetase (TyrRS) is involved in various functions including protection from DNA damage due to oxidative stress, protein synthesis and cell signaling and can be an attractive target for controlling the pathogens by early inhibition of translation. TyrRS has two disordered regions, which lack a stable 3D structure in solution, and are involved in tRNA synthetase catalysis and stability. One of the disordered regions undergoes disorder-to-order transition (DOT) upon complex formation with tRNA whereas the other remains disordered (DR). In this work, we have explored the importance of these disordered regions using molecular dynamics simulations of both free and RNA-complexed states. We observed that the DOT and DR regions of the first subunit acts as a flap and interact with the acceptor arm of the tRNA. The DOT-DR flap closes when tyrosine (TyrRSTyr) is present at the active site of the complex and opens in the presence of tyrosine monophosphate (TyrRSYMP). The DOT and DR regions of the second subunit interact with the anticodon stem as well as D-loop of the tRNA, which might be involved in stabilizing the complex. The anticodon loop of the tRNA binds to the structured region present in the C-terminal of the protein, which is observed to be flexible during simulations. Detailed energy calculations also show that TyrRSTyr complex has stronger binding energy between tRNA and protein compared to TyrRSYMP; on the contrary, the anticodon is strongly bound in TyrRSYMP. The results obtained in the present study provide additional insights for understanding catalysis and the involvement of disordered regions in Tyr transfer to cognate tRNA.

An Orthogonal Tyrosyl-tRNA Synthetase/tRNA Pair from a Thermophilic Bacterium for an Expanded Eukaryotic Genetic Code.

The Escherichia coli-derived tyrosyl-tRNA synthetase was the first enzyme engineered for genetic code expansion in a eukaryotic system but can charge only a limited set of structurally simple noncanonical amino acids. In contrast, the thermophilic Methanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase mutants, used in only a prokaryotic system, can charge a surprisingly large set of structurally diverse ncAAs, due to their remarkable structural ability to tolerate mutations. Inspired by this, we characterized a new class of tyrosyl-tRNA synthetase/tRNATyr pairs from thermophilic bacterium Geobacillus stearothermophilus, which is homologous to the E. coli tyrosyl-tRNA synthetase but with better thermostability. This new pair is both orthogonal in mammalian cells and in Saccharomyces cerevisiae for genetic code expansion and can charge a diverse set of ncAAs with a comparable cellular efficiency, better specificity, and lower background, as compared to those of its E. coli homologue. This thermostable enzyme provides an alternative scaffold for synthetase library screening or evolution to genetically encode more structurally complex ncAAs in eukaryotic cells.

A bisubstrate reagent orchestrating adenosine triphosphate and l-tyrosine and making tyrosyl adenylate: partial mimicking of tyrosyl-tRNA synthetase.

We report the development of a bisubstrate reagent that, similar to tyrosyl t-RNA synthetase (TyrTS), provides a surface for ATP and l-Tyr to render a pseudo-intramolecular reaction forming 5'-tyrosyl adenylate (tyrAd). The presence of the reagent in solution with TyrTS marred the enzymatic reaction and, noticeably, tyrAd formed under the catalytic mode of the biomodel reagent was not picked up by TyrTS and hence was not transferred to tRNA. A potential application of this reagent, which doesn't allow the formation of tyrosyl tRNA, may lie in an emerging therapeutic targeting the translation machinery of cells without inhibiting the normal workings of enzymes.

Cell-Free Expression of Unnatural Amino Acid Incorporated Aquaporin SS9 with Improved Separation Performance in Biomimetic Membranes.

Aquaporins (AQPs) are widely applied in biomimetic membranes for water recycling and desalination. In this study, a novel aquaporin was isolated from Photobacterium profundum SS9 (AQP SS9), which showed high water permeability and potential for practical water purification applications. To improve the stability of the AQP SS9 embedded biomimetic membranes, a modified AQP SS9 was obtained by incorporation of an unnatural amino acid (p-propargyloxyphenylalanine, pPpa) (P-AQP SS9) in vitro using a mutated Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and the cell-free expression system. The modified AQP SS9 can covalently link with phospholipids and hence significantly improve the stability of biomimetic membranes. The concentration of Mg2+ and fusion expression with signal peptides were evaluated to enhance the expression level of P-AQP SS9, resulting in a highest yield of 49 mg/L. The modified AQP SS9 was then reconstituted into DOPC liposomes and analyzed by a stopped-flow spectrophotometer. The obtained water permeability coefficient (Pf) of 7.46×10-4 m/s was 5.7 times higher than that of proteoliposomes with the wild-type AQP SS9 (Pf=1.31×10-4 m/s) and 12.1 times higher than that of the DOPC liposomes (Pf=6.15×10-5m/s). This study demonstrates the development of a cell-free system for the expression of membrane proteins with much higher stability and the potential application of the modified aquaporins for water filtration.

A noncanonical amino acid-based relay system for site-specific protein labeling.

Genetically site-specific introduction of noncanonical amino acids (ncAAs) for protein conjugation generally requires incorporation through exogenous feeding of chemically synthesized ncAAs. We developed a p-amino-phenylalanine (pAF)-based relay system that enables site-specific functionalization of proteins without chemical synthesis of the building blocks. pAF was biosynthesized under optimized conditions, followed by site-specific incorporation into a specific protein residue. The resulting protein was ready for functionalization using an oxidative conjugation reaction. We demonstrated the use of this relay system by preparing a fluorophore-labeled anti-HER2 single-chain variable fragment antibody for fluorescent imaging.

The genetic incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast.

The noncanonical amino acid p-azidomethyl-l-phenylalanine can be genetically incorporated into proteins in bacteria, and has been used both as a spectroscopic probe and for the selective modification of proteins by alkynes using click chemistry. Here we report identification of Escherichia coli tyrosyl tRNA synthetase mutants that allow incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast. When expressed together with the cognate E. coli tRNACUATyr, the new mutant tyrosyl tRNA synthetases directed robust incorporation of p-azidomethyl-l-phenylalanine into a model protein, human superoxide dismutase, in response to the UAG amber nonsense codon. Mass spectrometry analysis of purified superoxide dismutase proteins confirmed the efficient site-specific incorporation of p-azidomethyl-l-phenylalanine. This work provides an additional tool for the selective modification of proteins in eukaryotic cells.

Resveratrol's Hidden Hand: A Route to the Optical Detection of Biomolecular Binding.

Resveratrol is a stilbenoid phytoalexin with promising myriad health benefits predominantly contributed by the trans ( E) diastereomeric form. A recent study has implicated the cis ( Z) diastereomer in human health. This stereoisomer binds with high affinity to human tyrosyl-tRNA synthetase, initiating a downstream cascade that promotes the expression of genes associated with the cellular stress response. We discovered that the nonplanar structure of the cis-resveratrol conformer possesses certain chiral signals in its simulated vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectra. These features may be used for the optical detection of the binding event and in understanding the more diversified biological roles of trans-resveratrol over cis-resveratrol. We use a density functional theory model, which is validated against the known results for the E diastereomer. The Z diastereomer is significantly nonplanar and can exist in two helical atropisomeric forms. These forms exchange rapidly in solution, but only one is observed to bind with the synthetase. This suggests that the binding may generate an enantiomeric excess, leading to detectable changes in the vibrational optical activity spectra. We identify candidate features at 998, 1649, and 1677 cm-1 in the ROA and at 1642 and 3834 cm-1 in the VCD spectra of Z-resveratrol that may be useful for this purpose.