RESUMEN
A new method has been developed to reassign the rare codon AGA in Escherichia coli by engineering an orthogonal tRNA/aminoacyl-tRNA synthetase pair derived from Methanocaldococcus jannaschii. The tRNA mutant was introduced with a UCU anticodon, and the synthetase was evolved to correctly recognize the modified tRNA anticodon loop and to selectively charge a target noncanonical amino acid (NAA) onto the tRNA. In order to maximize the efficiency of AGA codon reassignment, while avoiding the lethal effects caused by global codon reassignment in cellular proteins, an inducible promoter (araBAD) was utilized to provide temporal controls for overexpression of the aminoacyl-tRNA synthetase and switch on codon reassignment. Using this system, we were able to efficiently incorporate p-acetylphenylalanine, O-methyl-tyrosine, and p-iodophenylalanine into proteins in response to AGA codons. Also, we found that E.â coli strain GM10 was optimal in achieving the highest AGA reassignment rates. The successful reassignment of AGA codons reported here provides a new avenue to further expand the genetic code.
Asunto(s)
Aminoácidos/genética , Codón/genética , Escherichia coli/genética , Código Genético , Aminoácidos/metabolismo , Escherichia coli/metabolismo , Methanocaldococcus/enzimología , Tirosina-ARNt Ligasa/metabolismoRESUMEN
The 2-naphthol analogue of tyrosine, 2-amino-3-(6-hydroxy-2-naphthyl)propanoic acid (NpOH), has been genetically introduced into proteins in Escherichia coli . This is achieved through the directed evolution of orthogonal aminoacyl-tRNA synthetase/tRNA pairs that selectively charge the target amino acid in response to the amber stop codon, UAG. Moreover, chemoselective azo coupling reactions have been revealed between the 2-naphthol group and diazotized aniline derivatives that are substituted with an electron donating moiety. The coupling reactions required a very mild condition (pH 7) with great reaction rate (less than 2 h at 0 °C), high efficiency, and excellent selectivity.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Naftoles/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Evolución Molecular Dirigida/métodos , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Methanococcus/enzimología , Methanococcus/genética , Modelos Moleculares , Naftoles/química , Ingeniería de Proteínas/métodos , Tirosina/metabolismoRESUMEN
For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269-277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO(2)Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-alpha (mTNF-alpha) independently to pNO(2)Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-alpha IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-alpha and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO(2)Phe(43) mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.
Asunto(s)
Aminoácidos/genética , Inmunoterapia/métodos , Ingeniería de Proteínas/métodos , Autotolerancia/inmunología , Aminoácidos/inmunología , Animales , Formación de Anticuerpos , Autoanticuerpos , Autoantígenos/genética , Inmunoglobulina G , Ratones , Fenilalanina/análogos & derivados , Fenilalanina/genética , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V(H) CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded "synthetic" genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.
Asunto(s)
Evolución Molecular Dirigida , Código Genético , Proteínas/genética , Aminoácidos/genética , Anticuerpos Antivirales/inmunología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Vectores Genéticos , Células Germinativas/inmunología , VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Biblioteca de Péptidos , Análisis de Secuencia de Proteína , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-alpha) to p-nitrophenylalanine (pNO(2)Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) --> Phe mutant. The antibodies generated against the pNO(2)Phe are highly cross-reactive with native mTNF-alpha and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.
Asunto(s)
Sustitución de Aminoácidos , Formación de Anticuerpos/efectos de los fármacos , Mutación Missense , Autotolerancia/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Enfermedades Transmisibles/genética , Enfermedades Transmisibles/inmunología , Endotoxemia/inducido químicamente , Endotoxemia/tratamiento farmacológico , Endotoxemia/genética , Endotoxemia/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/farmacología , Inmunoquímica , Lipopolisacáridos/toxicidad , Masculino , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Nitrofenoles/inmunología , Nitrofenoles/farmacología , Autotolerancia/genética , Autotolerancia/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Vacunas/genética , Vacunas/inmunologíaRESUMEN
The unnatural amino acid p-nitrophenylalanine (pNO2-Phe) was genetically introduced into proteins in Escherichia coli in response to the amber nonsense codon with high fidelity and efficiency by means of an evolved tRNA/aminoacyl-tRNA synthetase pair from Methanocuccus jannaschii. It was shown that pNO2-Phe efficiently quenches the intrinsic fluorescence of Trp in a distance-dependent manner in a model GCN4 basic region leucine zipper (bZIP) protein. Thus, the pNO2-Phe/Trp pair should be a useful biophysical probe of protein structure and function.
Asunto(s)
Escherichia coli/genética , Proteínas Mutantes/genética , Fenilalanina/análogos & derivados , Proteínas Recombinantes/genética , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/metabolismo , Methanococcus/enzimología , Methanococcus/genética , Datos de Secuencia Molecular , Proteínas Mutantes/biosíntesis , Fenilalanina/genética , Fenilalanina/metabolismo , ARN de Transferencia de Tirosina/genética , ARN de Transferencia de Tirosina/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Factores de Transcripción/genética , Triptófano/química , Triptófano/genética , Triptófano/metabolismo , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismoRESUMEN
[reaction: see text] Photolysis of 3 in argon-saturated 2-propanol led to formation of 5 via intermolecular H-atom abstraction followed by lactonization. Irradiation of 4 in 2-propanol gave compounds 6 and 7 that also come from intermolecular H-atom abstraction. In contrast, photolysis of an oxygen-saturated solution of 3 in 2-propanol yields products 8, 9, and 10, which were all formed from intramolecular H-atom abstraction and trapping of the corresponding biradical with oxygen. Laser flash photolysis of 3 in methanol showed formation of biradical 3BR (lambda(max) 330 nm, and tau = 50 ns) via intramolecular H-atom abstraction as the main photoreactivity of 3. Biradical 3BR decayed into photoenols 3Z and 3E (lambda(max) 390 nm, tau = 6.5 micros and tau = 162 micros, respectively). In comparison, laser flash photolysis of 4 yielded photoenols 4Z and 4E (lambda(max) 390 nm, tau = 15 micros and tau = 3.6 ms, respectively). Thus photoenol 3E is unusually short-lived, and therefore it does not undergo the intramolecular lactonization as we have observed for the analogous photoenol 1E. Photoenol 3Z decays back to 3 via an intramolecular 1,5-H shift, whereas photoenol 3E reforms 3 efficiently via the solvent with the aid of the ortho ester group. The intramolecular lactonization of photoenols 1E and 3E must be a slow process, presumably because the photoenols are rigid and the hydroxyl group is inhibited, by intramolecular hydrogen bonding, from acquiring the correct geometry for lactonization. Thus only photoenols that are resistant to reformation of their ketone via the solvent are long-lived enough to undergo lactonization and release the alcohol moiety.
RESUMEN
The dynamics of singlet 3,5-dichloro-2-biphenylnitrene and the products of its rearrangement were monitored by pico- and nanosecond laser flash photolysis and the results were consistent with the predictions of DFT and ab initio molecular orbital calculations.
Asunto(s)
Azidas/análisis , Azidas/química , Compuestos de Bifenilo/química , Simulación por Computador , Bifenilos Policlorados/análisis , Rayos Láser , Estructura Molecular , Fotoquímica , Fotólisis , Bifenilos Policlorados/química , Temperatura , Factores de Tiempo , Rayos UltravioletaRESUMEN
We report a general method to display peptide-containing unnatural amino acids on filamentous M13 phage. Five distinct unnatural amino acids were site-specifically incorporated at the N-terminal of the M13 phage minor coat protein pIII. Phages that contain p-azidophenylalanine can undergo a highly specific azide-alkyne [3 + 2] cycloaddition reaction with an alkyne-derivatized fluorophore. The generalization of phage display to include unnatural amino acids should significantly increase the scope of phage display technology.
Asunto(s)
Aminoácidos/genética , Aminoácidos/metabolismo , Biosíntesis de Péptidos/genética , Biblioteca de Péptidos , Proteínas de la Cápside , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Methanococcus/genética , Methanococcus/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
Ar matrix photolysis of 1- and 2-naphthyl azides 3 and 4 at 313 nm initially affords the singlet naphthyl nitrenes, (1)()1 and (1)()2. Relaxation to the corresponding lower energy, persistent triplet nitrenes (3)()1 and (3)()2 competes with cyclization to the azirines 15 and 18, which can also be formed photochemically from the triplet nitrenes. On prolonged irradiation, the azirines can be converted to the seven-membered cyclic ketenimines 10 and 13, respectively, as described earlier by Dunkin and Thomson. However, instead of the o-quinoid ketenimines 16 and 19, which are the expected primary ring-opening products of azirines 15 and 18, respectively, we observed their novel bond-shift isomers 17 and 20, which may be formally regarded as cyclic nitrile ylides. The existence of such ylidic heterocumulenes has been predicted previously, but this work provides the first experimental observation of such species. The factors which are responsible for the special stability of the ylidic species 17 and 20 are discussed.
RESUMEN
Phenyl azide, 2,6-diethylphenyl azide, 2,6-diisopropylphenyl azide, and 2,4,6-tri-tert-butylphenyl azide were studied by laser flash photolysis (LFP) methods. LFP (266 nm) of the azides in glassy 3-methylpentane at 77 K produces the transient UV-vis absorption spectra of the corresponding singlet nitrenes. At 77 K, the singlet nitrenes relax to the corresponding triplet nitrenes. The triplet nitrenes are persistent at 77 K and their spectra were recorded. The rate constants of singlet to triplet intersystem crossing were determined at this temperature. LFP of 2,4,6-tri-tert-butyl phenyl azide in pentane at ambient temperature again produces a singlet nitrene, which is too short-lived to detect by nanosecond spectroscopy under these conditions. Unlike the other azides, the first detectable intermediate produced upon LFP of 2,4,6-tri-tert-butyl phenyl azide at ambient temperature is the benzazirine (285 nm) which has a lifetime of 62 ns controlled by ring opening to a didehydroazepine. The results are interpreted with the aid of Density Functional Theoretical and Molecular Orbital Calculations.
RESUMEN
The photochemistry of ortho-biphenyl azide (1a) has been studied by laser flash photolysis (LFP), with UV-vis and IR detection of the transient intermediates formed. LFP (266 nm) of 1a in glassy 3-methylpentane at 77 K releases singlet ortho-biphenylnitrene (1b) (lambda(max) = 410 nm, tau = 59 +/- 6 ns), which under these conditions decays cleanly to the lower energy triplet state. In fluid solution at 298 K, 1b rapidly (tau < 10 ns) partitions between formation of isocarbazole (4) (lambda(max) = 430 nm, tau = 70 ns) and benzazirine (1e) (lambda(max) = 305 nm, tau = 12 ns). Isocarbazole 4 undergoes a 1,5-hydrogen shift, with k(H)/k(D) = 3.4 at 298 K to form carbazole 9 and smaller amounts of two other isocarbazoles (7 and 8). Benzazirine 1e ring-opens reversibly to azacycloheptatetraene (1f), which serves as a reservoir for singlet nitrene 1b. Azacycloheptatetraene 1f ultimately forms carbazole 9 on the millisecond time scale by the pathway 1f --> 1e --> 1b --> 4 --> 9. The energies of the transient intermediates and of the transition structures connecting them were successfully predicted by CASPT2/6-31G calculations. The electronic and vibrational spectra of the intermediates, computed by density functional theory, support the assignment of the transient spectra, observed in the formation of 9 from 1a.
RESUMEN
The structures of halogen atom-benzene complexes were investigated by modern DFT and ab initio computational methods. The spectroscopic properties of the complexes are also predicted and are in good agreement with experiment where such data have been reported. The fluorine atom-benzene complex is predicted to be a sigma complex due to the strength of a C-F bond. The chlorine atom-benzene complex is predicted to have an eta(1) pi complex structure, which is only slightly more favorable (1.1 kcal/mol with the BH&HLYP/6-311++G method including the ZPE correction) than a sigma complex but is significantly more stable (4.4 kcal/mol with the BH&HLYP/6-311++G method including the ZPE correction) than the eta(6) pi complex. The bromine and iodine benzene complexes are also predicted to prefer an eta(1) pi complex structure.
RESUMEN
As a model for riboflavin, lumiflavin was investigated using density functional theory methods (B3LYP/6-31G* and B3LYP/6-31+G**) with regard to the proposed cascade of intermediates formed after excitation to the triplet state, followed by electron-transfer, proton-transfer, and radical[bond]radical coupling reactions. The excited triplet state of the flavin is predicted to be 42 kcal/mol higher in energy than the singlet ground state, and the pi radical anion lies 45.1 kcal/mol lower in energy than the ground-state flavin and a free electron in the gas phase. The former value compares to a solution-phase triplet energy of 49.8 kcal/mol of riboflavin. For the radical anion, the thermodynamically favored position to accept a proton on the flavin ring system is at N(5). A natural population analysis also provided spin density information for the radicals and insight into the origin of the relative stabilities of the six different calculated hydroflavin radicals. The resulting 5H-LF* radical can then undergo radical[bond]radical coupling reactions, with the most thermodynamically stable adduct being formed at C(4'). Vibrational spectra were also calculated for the transient species. Experimental time-resolved infrared spectroscopic data obtained using riboflavin tetraacetate are in excellent agreement with the calculated spectra for the triplet flavin, the radical anion, and the most stable hydroflavin radical.