RESUMO
A small and highly fluorescent non-natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four-base codon/anticodon pair. The non-natural mutants were obtained in excellent yields and some of them retained strong biotin-binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non-natural amino acid for a position-specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.
Assuntos
Aminoácidos/química , Aminoácidos/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Proteínas/química , ortoaminobenzoatos/química , ortoaminobenzoatos/síntese química , Sítios de Ligação/genética , Biotina , Escherichia coli/genética , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Fluorescência , Estreptavidina/biossíntese , Estreptavidina/química , Estreptavidina/genéticaRESUMO
Incorporation of nonnatural amino acids into proteins is a powerful technique in protein research. Amber suppression has been used to this end, but this strategy does not allow multiple incorporation of nonnatural amino acids into single proteins. In this article, we developed an alternative strategy for nonnatural mutagenesis by using four-base codons. The four-base codons AGGU, CGGU, CCCU, CUCU, CUAU, and GGGU were successfully decoded by the nitrophenylalanyl-tRNA containing the complementary four-base anticodons in an Escherichia coli in vitro translation system. The most efficient four-base decoding was observed for the GGGU codon, which yielded 86% of the full-length protein containing nitrophenylalanine relative to the wild-type protein. Moreover, highly efficient incorporation of two different nonnatural amino acids was achieved by using a set of two four-base codons, CGGG and GGGU. This work shows that the four-base codon strategy is more advantageous than the amber suppression strategy in efficiency and versatility.
Assuntos
Aminoácidos/genética , Códon , Mutagênese , Biossíntese de Proteínas , Anticódon , Sistema Livre de Células , Escherichia coli , Conformação de Ácido Nucleico , Aminoacil-RNA de Transferência/metabolismoRESUMO
Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon-anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA(UACCG) chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN(1)N(2), where N(1) and N(2) indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.
Assuntos
Aminoácidos/genética , Proteínas de Bactérias/genética , Códon/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Anticódon/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Western Blotting , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Mutação , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA de Transferência de Tirosina/genética , Estreptavidina/química , Estreptavidina/genética , Estreptavidina/metabolismo , Tirosina/genéticaRESUMO
Oxy-peptide nucleic acids (OPNAs) of [-NH-CH(CH2-CH2-Base)-CH2-O-CH2-CO-]-type main chain with four different types of nucleobases (Base = A, G, C, and U) or with an abasic side group (X) were synthesized. Melting curves of the 1:1 hybrids of o(A(n))-d(Tn)) pairs with n = 6, 9, 12, and 15 showed very sharp transitions at high Tm values, particularly for long chains, indicating that nearly optimum matching is attained in the structure of the o(A(n))-d(Tn) hybrids. Effect of different types of base pairs on the hybrid stabilities was examined for the o(A4NA4)-d(T4N'T4) 1:1 mixtures where N is A, G, C, U, or X and N' is A, G, C, or T. In all series of the hybrids the complementary pairs showed the highest Tm values. The Tm values of the complementary pairs were about 35 degrees C when purine bases were inserted as the N group in the OPNA, but they were 20-23 degrees C when pyrimidine bases were inserted. The melting curves of the hybrids with a single mismatch were similar to those with a single X-N' pair, suggesting that the mismatch base pairs have been ignored in the hybrids. All complementary OPNA-DNA hybrids showed higher Tm values and sharper transitions than the corresponding DNA-DNA hybrids. The OPNA-DNA hybrids favor a parallel direction i.e., the N-terminal of OPNA is directed to the 5'-terminal of DNA.
Assuntos
DNA/química , Hibridização de Ácido Nucleico/métodos , Oligopeptídeos/química , Ácidos Nucleicos Peptídicos/química , Pareamento de Bases , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , DNA Complementar/química , Oligopeptídeos/síntese química , Ácidos Nucleicos Peptídicos/síntese química , TemperaturaRESUMO
Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.
Assuntos
Anticódon/química , Anticódon/metabolismo , Códon/química , Proteínas/síntese química , Proteínas/genética , Aminoácidos/química , Aminoácidos/genética , Anticódon/genética , Códon/genética , Mutagênese , RNA de Transferência/metabolismoRESUMO
The interaction of antisense peptide nucleic acid (PNA) with yeast tRNA(Phe) was investigated. A 6-mer PNA complementary to the 3'-terminal sequence including the 73ACCA end hybridized to the tRNA. While the PNA with a single mismatch did not. PNA is a promising candidate for controlling tRNA functions by the sequence-specific hybridization.
Assuntos
Elementos Antissenso (Genética)/química , Ácidos Nucleicos Peptídicos/química , RNA de Transferência/química , Aminoacil-tRNA Sintetases/química , Elementos Antissenso (Genética)/farmacologia , Sequência de Bases , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/farmacologia , RNA de Transferência/fisiologiaRESUMO
Frameshift suppressor tRNAs containing an additional nucleotide at anticodon loop were prepared and their translational activity was evaluated in an E. coli in vitro translation system. The mutated tRNA(CCCG) was chemically aminoacylated with nitrophenylalanine, and added to the in vitro translation system together with a streptavidin mRNA containing CGGG codon. Western blot analysis indicates that the mutated tRNAs can decode the four-base codon, except 32.1C and 33.1G mutants.
Assuntos
Anticódon/química , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Mudança da Fase de Leitura do Gene Ribossômico , Dados de Sequência Molecular , Mutagênese Insercional , RNA de Transferência/química , RNA de Transferência/genéticaRESUMO
Site-specific incorporation of the tyrosine isostere (2-Hydroxy-3-(4-hydroxy-phenyl)-propionic acid) into streptavidin was accomplished by in vitro frameshift suppression of a CGGG 4-base mutation with a chemically acylated frameshift suppressor tRNA(cccg). The mutant, in which the backbone amide linkage is replaced by an ester linkage, is hydrolyzed under neutral condition. The cleavage rate of the protein backbone at a single-predetermined site was strongly depended on the incorporated position of the isostere.
Assuntos
Anticódon/química , Códon/química , Fenilpropionatos/química , Estreptavidina/química , Estreptavidina/genética , Biossíntese de Proteínas , Estreptavidina/metabolismo , Tirosina/químicaRESUMO
Cell-free protein synthesis, driven by a crude S30 extract from Escherichia coli, has been applied to the preparation of proteins containing unnatural amino acids at specific positions. We have developed methods for inactivating tRNA(Asp) and tRNA(Phe) within a crude E. coli tRNA by an antisense treatment and for digesting most of the tRNA within the S30 extract without essential damage to the ribosomal activity. In the present study, we applied these methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids. With 10 mM Mg(2+), the translation efficiency was higher than that with the other tested concentration, and the misreading efficiency was low. The protease mRNA was translated in the presence of an antisense DNA-treated tRNA mixture and 2-naphthylalanyl- and/or p-phenylazophenylalanyl-tRNA. The results suggest that a good portion of the translation products are substituted at all of the seven positions originally occupied by Asp or Phe.
Assuntos
Códon/genética , DNA Antissenso/metabolismo , Escherichia coli/metabolismo , Protease de HIV/química , Protease de HIV/genética , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Fenilalanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/análogos & derivados , Compostos Azo , Sequência de Bases , Sistema Livre de Células , DNA Antissenso/genética , Protease de HIV/biossíntese , Magnésio/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fenilalanina/análogos & derivados , Biossíntese de Proteínas , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Fenilalanina/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position. Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations.
Assuntos
Aminoácidos/química , Biotina/química , Corantes Fluorescentes/síntese química , Estreptavidina/química , Cumarínicos/química , Indicadores e Reagentes , Mutação , RNA de Transferência/metabolismo , Proteínas Recombinantes/biossíntese , Espectrometria de Fluorescência , Estreptavidina/genética , Triptofano/químicaRESUMO
A new method was developed for the generation of a library of mutant proteins that contained nonnatural amino acids. The method, "random insertion and deletion (RID) mutagenesis", is based on the deletion of an arbitrary number of bases at random positions and, at the same time, the insertion of an arbitrary sequence into the same position. By using this method, randomly selected three consecutive bases in the gene of green fluorescence protein (GFP) were replaced by a CGGT 4-base codon. When this DNA library was expressed in E. coli, about 80% of colonies lost the fluorescence. The non-fluorescent colonies were picked up and the genes were sequenced. Replacement of three consecutive bases by CGGT 4-base codon was found in two of the four mutated genes.
Assuntos
Mutagênese Insercional/métodos , Biblioteca de Peptídeos , Proteínas/genética , Deleção de Sequência , Aminoácidos/química , Sequência de Bases , Códon/genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
A novel strategy for the incorporation of nonnatural amino acids into proteins was developed by using five-base codon-anticodon pairs. The streptavidin mRNA containing five-base codon CGGUA and the chemically aminoacylated tRNA with five-base anticodon UACCG were prepared, and added into E. coli in vitro translation system. As a result, the nonnatural amino acid was successfully incorporated into desired position of the protein. Other five-base codons CGGN1N2, where N1 and N2 indicate one of four nucleotides, were also available for the incorporation of the nonnatural amino acid.
Assuntos
Aminoácidos/química , Anticódon/genética , Códon/genética , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Biossíntese de Proteínas , Engenharia de Proteínas , RNA Bacteriano/genética , RNA Mensageiro/genética , Estreptavidina/análogos & derivados , Estreptavidina/química , Estreptavidina/genéticaRESUMO
Extension of biochemical functions has been attempted by introducing nonatural amino acids and artificial nucleic acid analogs. Nonnatural amino acids have been linked to tRNAs and the amino-acylated tRNAs were added to E. coli in vitro protein synthesizing system to produce nonnatural mutant proteins. The positions of the nonnatural amino acids have been assigned by the 4-base codons, like CGGG and AGGU. The extended codons have been introduced at a specific position or at random positions on a DNA. In the latter case, a DNA library that contains a single 4-base codon at random positions can be obtained. The combination of these new techniques opens a way to the introduction of artificial functions to biochemical systems.
Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Sequência de Bases , Códon/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes , Mutagênese , Mutagênese Insercional , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Syntheses of N-Fmoc delta-amino acids with an ether linkage in the main chain and six different nucleobases on the side chain, Fmoc-NH-C*H(CH2-CH2-B)-CH2-O-CH2-COOH (B = N6-benzoyladenine, thymine, uracil, N-benzoylcytosine, guanine, and N2-isobutyrylguanine) are described. The delta-amino acids were prepared through 8-12 step synthesis starting from L-homoserine and could be linked together to form novel peptide nucleic acids (oxy-PNAs = OPNAs) by solid-phase peptide synthesis.
Assuntos
Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/síntese química , Indicadores e Reagentes , Oligopeptídeos/síntese química , Oligopeptídeos/química , Purinas , Pirimidinas , EstereoisomerismoRESUMO
A novel method of the in vitro incorporation of two nonnatural amino acids into proteins through extension of the genetic code was developed. The streptavidin mRNA containing AGGU and CGGG, and chemically aminoacylated tRNA(ACCU) and tRNA(CCCG) were prepared, then they were added into E. coli in vitro protein synthesizing system. As a result, two nonnatural amino acids were successfully incorporated into desired sites of streptavidin.
Assuntos
Código Genético , Proteínas/genética , RNA Mensageiro/genética , Aminoacil-RNA de Transferência/metabolismo , Estreptavidina/genética , Sequência de Bases , Códon/química , Escherichia coli/genética , Escherichia coli/metabolismo , Biossíntese de Proteínas , Proteínas/química , RNA Mensageiro/química , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genéticaRESUMO
Nonnatural amino acids with photofunctional groups were incorporated site-specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNA(CCU) through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. L-p-Phenylazophenylalanine, L-2-anthrylalanine, L-1-naphthylalanine, L-2-naphthylalanine and L-p-biphenylalanine were successfully incorporated into a polypeptide, but l-1-pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.
Assuntos
Alanina/análogos & derivados , Aminoácidos/metabolismo , Fenilalanina/análogos & derivados , Biossíntese de Proteínas , Acilação , Alanina/metabolismo , Antracenos/metabolismo , Compostos Azo/metabolismo , Sequência de Bases , Códon , Escherichia coli/metabolismo , Dados de Sequência Molecular , Fenilalanina/metabolismo , Fotoquímica , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Arginina/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , Espectrometria de FluorescênciaRESUMO
3'-N-Aminoacyl analogs of puromycin with nonnatural aromatic amino acids were synthesized and their inhibitory activity in E. coli in vitro protein synthesizing system was evaluated. The analogs with L-2-naphthylalanine, L-p-biphenylalanine, L-2-anthrylalanine and trans-L-p-phenylazophenylalanine were found to inhibit the protein synthesis with high efficiency. The inhibition suggests that these nonnatural amino acids are accepted by the active center of the E. coli ribosomal A site. A model for the adaptability of nonnatural aromatic amino acids to the active center is proposed.
Assuntos
Alanina/análogos & derivados , Escherichia coli/efeitos dos fármacos , Fenilalanina/análogos & derivados , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/análogos & derivados , Ribossomos/metabolismo , Alanina/metabolismo , Alanina/farmacologia , Proteínas de Bactérias/biossíntese , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Sítios de Ligação , Escherichia coli/metabolismo , Estrutura Molecular , Fenilalanina/metabolismo , Fenilalanina/farmacologia , Inibidores da Síntese de Proteínas/metabolismo , Puromicina/metabolismo , Puromicina/farmacologia , Ribossomos/efeitos dos fármacos , Raios UltravioletaRESUMO
A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.
Assuntos
Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo/efeitos da radiação , Compostos Azo/efeitos da radiação , Animais , Compostos Azo/imunologia , Cromatografia Líquida de Alta Pressão , Fluorescência , Haptenos/imunologia , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Raios UltravioletaRESUMO
Novel polyetherurethaneureas which have been synthesized by the present authors were chosen for the substrate polymers, on which adhesion of platelets was investigated. The number of adhered platelets and the amount of serotonin released from platelets adhered on the polymers and the protein-coated polymers were determined by radioisotope method. Both of them were enhanced with increasing content of urea linkages in the polyetherurethaneureas. The platelet adhesion was discussed in terms of the denaturation of plasma proteins upon adsorption, which was determined by Fourier-transform infrared spectroscopy. With increasing degree of protein denaturation, the platelet adhesion and the serotonin release were enhanced. This relationship was particularly evident in the case of albumin adsorption. It was shown that the surface properties of substrate polymers affect the protein adsorption, which in turn influences the adhesion of platelets.
Assuntos
Materiais Biocompatíveis , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Adesividade Plaquetária , Poliuretanos , Adsorção , Animais , Cães , Desnaturação Proteica , Serotonina/metabolismoRESUMO
Interactions of platelet with novel polyetherurethaneurea and its heparinized derivative were investigated. Platelet adhesion onto the material and release of serotonin or adenosine phosphate from platelet-rich plasma (PRP) were suppressed by an introduction of amino groups to polyetherurethaneurea, by quaternization of the polymer, and further by heparinization of the polymer. When the material was precoated with one of major plasma proteins and the protein-coated materials were taken to contact with washed platelet suspension (WP), the dependence of platelet adhesion and activation on the properties of polymers was different from that observed for PRP interaction. Platelet adhesion and activation were promoted according to the nature of coating proteins in the order albumin less than gamma-globulin less than fibrinogen and with increasing degree of denaturation of coating proteins. When the polymer materials were coated with proteins by immersing in aqueous solution containing two kinds of plasma proteins, adhesion behaviors of platelet were similar to those observed for PRP-uncoated material interaction. These experimental facts indicate that the selectivity of platelet for protein-coated material cannot be assessed by the interaction of WP with materials coated with a single kind of protein. It was concluded that material surface to which albumin is selectively adsorbed without denaturation does not stimulate adhering platelets for release reactions.