RESUMO
We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne-bearing pyrrolysine amino acid was biosynthesized and incorporated site-specifically allowing orthogonal double protein labeling.
Assuntos
Anidrases Carbônicas/metabolismo , Lisina/análogos & derivados , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Vias Biossintéticas , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Biossíntese de ProteínasRESUMO
Subtilosin A is a 35-residue, ribosomally synthesized bacteriocin encoded by the sbo-alb operon of Bacillus subtilis. It is composed of a head-to-tail circular peptide backbone that is additionally restrained by three unusual thioether bonds between three cysteines and the α-carbon of one threonine and two phenylalanines, respectively. In this study, we demonstrate that these bonds are synthesized by the radical S-adenosylmethionine enzyme AlbA, which is encoded by the sbo-alb operon and comprises two [4Fe-4S] clusters. One [4Fe-4S] cluster is coordinated by the prototypical CXXXCXXC motif and is responsible for the observed S-adenosylmethionine cleavage reaction, whereas the second [4Fe-4S] cluster is required for the generation of all three thioether linkages. On the basis of the obtained results, we propose a new radical mechanism for thioether bond formation. In addition, we show that AlbA-directed substrate transformation is leader-peptide dependent, suggesting that thioether bond formation is the first step during subtilosin A maturation.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Peptídeos Cíclicos/metabolismo , Sulfetos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Bacteriocinas/química , Sequência de Bases , Sítios de Ligação , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Ferro-Enxofre/genética , Dados de Sequência Molecular , Mutagênese , Óperon , Peptídeos Cíclicos/química , Fenilalanina/química , S-Adenosilmetionina/metabolismo , Treonina/químicaRESUMO
Three for two: by using a Methanosarcina mazei PylRS triple mutant (Y306G, Y384F, I405R) the incorporation of two new exo-norbornene-containing pyrrolysine analogues was achieved. X-ray crystallographic analysis led to the identification of the crucial structural elements involved in substrate recognition by the evolved synthetase.
Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Norbornanos/química , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Química Click , Cristalografia por Raios X , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Methanosarcina/enzimologia , Mutação , Estrutura Terciária de ProteínaRESUMO
Significant differences in the reactivity of norbornene derivatives in the inverse electron-demand Diels-Alder reaction with tetrazines were revealed by kinetic studies. Substantial rate enhancement for the exo norbornene isomers was observed. Quantum-chemical calculations were used to rationalize and support the observed experimental data.
Assuntos
Anidridos/química , Norbornanos/química , Reação de Cicloadição , Elétrons , Cinética , Estrutura Molecular , Teoria QuânticaRESUMO
Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.
Assuntos
Imunoconjugados , Pró-Fármacos , Receptores de Glucocorticoides , Inibidores do Fator de Necrose Tumoral , Anticorpos , Pró-Fármacos/farmacologia , Glucocorticoides , Maleimidas , Imunoconjugados/farmacologiaRESUMO
We describe a new bioconjugation reaction based on the aziridination of norbornenes using electron-deficient sulfonyl azides. The reaction enables to attach various useful tags to peptides and proteins under mild conditions.
Assuntos
Azidas/química , Aziridinas/química , Norbornanos/química , Peptídeos/química , Proteínas/química , Sequência de Aminoácidos , Modelos Moleculares , Coloração e Rotulagem , Compostos de Enxofre/químicaRESUMO
Recently new lysine modifications were detected in histones and other proteins. Using the pyrrolysine amber suppression system we genetically inserted three of the new amino acids ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine site specifically into histone H3. The lysine at position 9 (H3 K9), which is known to be highly modified in chromatin, was replaced by these unnatural amino acids.