RESUMO
The unsaturated amino acid 2-amino-3-methyl-4-pentenoic acid (E-Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS-E-Ile and SR-E-Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS-E-Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %; SR-E-Ile yielded no conclusive evidence for incorporation. ATP/PPi exchange assays indicated that SS-E-Ile was activated by the isoleucyl-tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR-E-Ile was activated approximately 100-times more slowly than SS-E-Ile.
Assuntos
Escherichia coli/metabolismo , Isoleucina/química , Isoleucina/metabolismo , Ácidos Pentanoicos/química , Ácidos Pentanoicos/metabolismo , Tetra-Hidrofolato Desidrogenase/biossíntese , Tetra-Hidrofolato Desidrogenase/química , Animais , Escherichia coli/enzimologia , Escherichia coli/genética , Isoleucina/análogos & derivados , Isoleucina-tRNA Ligase/química , Isoleucina-tRNA Ligase/metabolismo , Cinética , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Camundongos , Conformação Molecular , Ácidos Pentanoicos/síntese química , Padrões de Referência , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estereoisomerismo , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
Methods for engineering proteins that contain non-canonical amino acids have advanced rapidly in the past few years. Novel amino acids can be introduced into recombinant proteins in either a residue-specific or site-specific fashion. The methods are complementary: residue-specific incorporation allows engineering of the overall physical and chemical behavior of proteins and protein-like macromolecules, whereas site-specific methods allow mechanistic questions to be probed in atomistic detail. Challenges remain in the engineering of the translational apparatus and in the design of schemes that can be used to encode both canonical and non-canonical amino acids.
