RESUMEN
Activated esters are widely used to label proteins at lysine side chains and Nâ termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins.
Asunto(s)
Lisina/metabolismo , Proteínas/metabolismo , Cristalografía por Rayos X , Teoría Funcional de la Densidad , Humanos , Conformación Proteica , Proteínas/química , TermodinámicaRESUMEN
Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.
Asunto(s)
Anticuerpos Monoclonales/química , Coenzima A/química , Citotoxinas/química , Inmunoconjugados/química , Aminobenzoatos/administración & dosificación , Aminobenzoatos/química , Animales , Citotoxinas/administración & dosificación , Estabilidad de Medicamentos , Ratones , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Relación Estructura-ActividadRESUMEN
Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.
Asunto(s)
Aminobenzoatos/metabolismo , Antineoplásicos/metabolismo , Proteínas Bacterianas/metabolismo , Inmunoconjugados/metabolismo , Neoplasias/tratamiento farmacológico , Oligopéptidos/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Trastuzumab/metabolismo , Aminobenzoatos/química , Aminobenzoatos/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Humanos , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Péptidos/química , Péptidos/metabolismo , Especificidad por Sustrato , Trastuzumab/química , Trastuzumab/uso terapéuticoRESUMEN
To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FcεRI, which is a key event in allergic disease.
Asunto(s)
Colorantes Fluorescentes/química , Lisina/análogos & derivados , Péptidos/química , Proteínas/química , Coloración y Etiquetado/métodos , Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Lisina/química , Estructura MolecularRESUMEN
The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.
Asunto(s)
Aminoácidos/inmunología , Linfocitos T CD4-Positivos/inmunología , Tolerancia Inmunológica/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Sustitución de Aminoácidos , Aminoácidos/genética , Animales , Autoanticuerpos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/inmunología , Factor de Crecimiento Epidérmico/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Inmunización/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Fenilalanina/análogos & derivados , Fenilalanina/genética , Fenilalanina/inmunología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Tirosina/inmunologíaRESUMEN
Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.
Asunto(s)
Lisina/química , Proteínas/química , Pirroles/química , Medios de Cultivo , Escherichia coli/genética , Resonancia Magnética Nuclear BiomolecularRESUMEN
D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.
Asunto(s)
Lisina/análogos & derivados , Ornitina/farmacología , Secuencia de Aminoácidos , Escherichia coli , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Lisina/biosíntesis , Lisina/química , Methanosarcina/genética , Methanosarcina/metabolismo , Estructura Molecular , Ornitina/química , Ornitina/metabolismo , Plásmidos , Regiones Promotoras GenéticasRESUMEN
Coronaviruses have been the causative agent of three epidemics and pandemics in the past two decades, including the ongoing COVID-19 pandemic. A broadly-neutralizing coronavirus therapeutic is desirable not only to prevent and treat COVID-19, but also to provide protection for high-risk populations against future emergent coronaviruses. As all coronaviruses use spike proteins on the viral surface to enter the host cells, and these spike proteins share sequence and structural homology, we set out to discover cross-reactive biologic agents targeting the spike protein to block viral entry. Through llama immunization campaigns, we have identified single domain antibodies (VHHs) that are cross-reactive against multiple emergent coronaviruses (SARS-CoV, SARS-CoV-2, and MERS). Importantly, a number of these antibodies show sub-nanomolar potency towards all SARS-like viruses including emergent CoV-2 variants. We identified nine distinct epitopes on the spike protein targeted by these VHHs. Further, by engineering VHHs targeting distinct, conserved epitopes into multi-valent formats, we significantly enhanced their neutralization potencies compared to the corresponding VHH cocktails. We believe this approach is ideally suited to address both emerging SARS-CoV-2 variants during the current pandemic as well as potential future pandemics caused by SARS-like coronaviruses.
Asunto(s)
COVID-19 , Camélidos del Nuevo Mundo , Anticuerpos de Dominio Único , Humanos , Animales , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Pandemias , EpítoposRESUMEN
Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.
Asunto(s)
Lisina/análogos & derivados , Proteínas/química , Proteínas/aislamiento & purificación , Benzaldehídos/química , Sitios de Unión , Lisina/química , Lisina/metabolismo , Estructura MolecularRESUMEN
The SARS-CoV-2 pandemic and particularly the emerging variants have deepened the need for widely available therapeutic options. We have demonstrated that hexamer-enhancing mutations in the Fc region of anti-SARS-CoV IgG antibodies lead to a noticeable improvement in IC50 in both pseudo and live virus neutralization assay compared to parental molecules. We also show that hexamer-enhancing mutants improve C1q binding to target surface. To our knowledge, this is the first time this format has been explored for application in viral neutralization and the studies provide proof-of-concept for the use of hexamer-enhanced IgG1 molecules as potential anti-viral therapeutics.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Inmunoglobulina G/genética , Pruebas Inmunológicas , Pandemias , SARS-CoV-2/genéticaRESUMEN
The solution structures of two different DNA duplexes (one containing a G-T mismatched base pair and the other a non-hydrogen-bonding G-F pair, where F is difluorotoluene) in complex with the peptide antibiotic actinomycin D (ActD) are presented. Using (1)H, (19)F NMR, and molecular dynamics simulations, we show that there are three major differences between the complexes: (1) ActD binds to the GF duplex in an orientation that is flipped 180° relative to its position in the GT duplex; (2) whereas the difluorotoluene moiety takes the typical anti glycosidic conformation in the "free" (uncomplexed) GF duplex, it takes the syn conformation in the GF:ActD complex; and (3) in GF:ActD, the difluorotoluene moiety is completely unstacked in the helix; however, the guanine of the G-F pair is stacked quite well with the ActD intercalator and the flanking base on the 5' side. In GT:ActD, the G-T base pair (although pushed into the major groove from the non-Watson-Crick hydrogen-bonding pattern) stacks favorably with the ActD intercalator and the flanking base pair on the 5' side. The results described here indicate that a sequence-specific DNA binding ligand such as actinomycin D will, indeed, recognize and bind with high affinity to a DNA incorporating a non-natural, non-hydrogen-bonding nucleoside mimic despite the presentation of modified functionality in the binding site.
Asunto(s)
Antibacterianos/metabolismo , ADN/química , ADN/metabolismo , Dactinomicina/metabolismo , Antibacterianos/química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Dactinomicina/química , Enlace de Hidrógeno , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido NucleicoRESUMEN
A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.
Asunto(s)
Aminoácidos/química , Marcaje Isotópico/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Proteínas/química , Isótopos de Carbono/química , Codón de Terminación , Flúor/química , Isótopos de Nitrógeno/química , Células Procariotas/química , Marcadores de SpinRESUMEN
We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser) in Saccharomyces cerevisiae in response to the amber nonsense codon TAG. This amino acid was converted to serine in living cells by irradiation with relatively low-energy blue light and was used to noninvasively photoactivate phosphorylation of the transcription factor Pho4, which controls the cellular response to inorganic phosphate. When substituted at phosphoserine sites that control nuclear export of Pho4, blocks phosphorylation and subsequent export by the receptor Msn5 (ref. 2). We triggered phosphorylation of individual serine residues with a visible laser pulse and monitored nuclear export of Pho4-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Pho4 mutants, which demonstrates dynamic regulation of Pho4 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Nitrobencenos/química , Nitrobencenos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/análogos & derivados , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Estructura Molecular , Mutación/genética , Fosforilación , Fotoquímica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Serina/química , Serina/genética , Serina/metabolismo , Factores de Transcripción/genéticaRESUMEN
A small molecule inhibitor of alpha4 integrin-dependent cell migration was identified through a cell-based screen of small molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with gamma-parvin. This molecule should serve as a useful tool to study alpha4 integrin signaling and may lead to new therapeutics for the treatment of autoimmune diseases.
Asunto(s)
Compuestos de Anilina/farmacología , Movimiento Celular/efectos de los fármacos , Integrina alfa4/metabolismo , Tubercidina/análogos & derivados , Actinina/antagonistas & inhibidores , Actinina/metabolismo , Adenosina Quinasa/antagonistas & inhibidores , Adenosina Quinasa/metabolismo , Compuestos de Anilina/síntesis química , Compuestos de Anilina/química , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Humanos , Integrina alfa4/efectos de los fármacos , Células Jurkat , Interferencia de ARN , Transducción de Señal , Bibliotecas de Moléculas Pequeñas , Tubercidina/síntesis química , Tubercidina/química , Tubercidina/farmacologíaRESUMEN
A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.
Asunto(s)
Aminoácidos/biosíntesis , Aminoácidos/genética , Aminoacil-ARNt Sintetasas/metabolismo , Código Genético , Lisina/análogos & derivados , Proteínas/química , Aminoácidos/química , Aminoacil-ARNt Sintetasas/genética , Animales , Archaea/genética , Células CHO , Línea Celular , Cricetinae , Cricetulus , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Biosíntesis de Proteínas , Proteínas/genéticaRESUMEN
4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.
Asunto(s)
Anticuerpos/química , Proteínas Bacterianas/química , Inmunoconjugados/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Antineoplásicos/química , Catálisis , Estructura Molecular , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
A protein evolution strategy is described by which double-stranded DNA fragments encoding defined Escherichia coli protein secondary structural elements (alpha-helices, beta-strands, and loops) are assembled semirandomly into sequences comprised of as many as 800 amino acid residues. A library of novel polypeptides generated from this system was inserted into an enhanced green fluorescent protein (EGFP) fusion vector. Library members were screened by fluorescence activated cell sorting (FACS) to identify those polypeptides that fold into soluble, stable structures in vivo that comprised a subset of shorter sequences ( approximately 60 to 100 residues) from the semirandom sequence library. Approximately 108 clones were screened by FACS, a set of 1149 high fluorescence colonies were characterized by dPCR, and four soluble clones with varying amounts of secondary structure were identified. One of these is highly homologous to a domain of aspartate racemase from a marine bacterium (Polaromonas sp.) but is not homologous to any E. coli protein sequence. Several other selected polypeptides have no global sequence homology to any known protein but show significant alpha-helical content, limited dispersion in 1D nuclear magnetic resonance spectra, pH sensitive ANS binding and reversible folding into soluble structures. These results demonstrate that this strategy can generate novel polypeptide sequences containing secondary structure.
Asunto(s)
Fragmentos de Péptidos/química , Biblioteca de Péptidos , Pliegue de Proteína , Proteínas/síntesis química , Evolución Molecular Dirigida , Proteínas de Escherichia coli , Estructura Secundaria de Proteína , SolubilidadRESUMEN
The incorporation of synthetic nucleoside analogues into DNA duplexes provides a unique opportunity to probe both structure and function of nucleic acids. We used 1H and 19F NMR and molecular dynamics calculations to determine the solution structures of two similar DNA decamer duplexes, one containing a central G-T mismatched or "wobble" base pair, and one in which the thymine in this base pair is replaced by difluorotoluene (a thymine isostere) creating a G-F pair. Here, we show that the non-hydrogen-bonding G-F pair stacks relatively well into the helix and that the distortions caused by each non-Watson-Crick G-T or G-F base pair are quite localized to a three base pair site around the mismatch. A detailed structural analysis reveals that the absence of hydrogen bonding introduces more dynamic motion into the G-F pair relative to G-T and permits the G-F pair to exhibit stacking and conformational features characteristic of both a Watson-Crick base pair (on the guanine containing strand) and a wobble base pair (on the strand containing the difluorotoluene). We used these results to posit a rationale for recognition and repair of mismatch sites in DNA.
Asunto(s)
ADN/química , Guanina/química , Tolueno/análogos & derivados , Emparejamiento Base , Reparación del ADN , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Teoría Cuántica , Soluciones , Tolueno/químicaRESUMEN
In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Fenilalanina/análogos & derivados , Fenilpropionatos/química , Proteínas/análisis , Tirosina/análogos & derivados , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Isótopos de Carbono , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graso Sintasas/química , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Marcaje Isotópico , Isótopos de Nitrógeno , Fenilpropionatos/metabolismo , Plásmidos/genética , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismoRESUMEN
Garlic's pungent flavor has made it a popular ingredient in cuisines around the world and throughout history. Garlic's health benefits have been elevated from folklore to clinical study. Although there is some controversy as to the efficacy of garlic, garlic products are one of the most popular herbal supplements in the U.S. Chemically complex, garlic contains different assortments of sulfur compounds depending on whether the cloves are intact, crushed, cooked, or raw. Raw garlic, when cut and placed on the tongue or lips, elicits painful burning and prickling sensations through unknown mechanisms. Here, we show that raw but not baked garlic activates TRPA1 and TRPV1, two temperature-activated ion channels that belong to the transient receptor potential (TRP) family. These thermoTRPs are present in the pain-sensing neurons that innervate the mouth. We further show that allicin, an unstable component of fresh garlic, is the chemical responsible for TRPA1 and TRPV1 activation and is therefore likely to cause garlic's pungency.