Assuntos
Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Cristalografia por Raios X , Dimetil Sulfóxido , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Fenilalanina/química , Plasmídeos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresAssuntos
Proteínas de Ligação ao Ferro/química , Enzimas de Conjugação de Ubiquitina/química , Isótopos de Carbono , Deutério , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas Mutantes/química , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Here we present an NMR-based approach to solving protein-ligand structures. The procedure is guided by biophysical, biochemical, or knowledge-based data. The structures are mainly derived from ligand-induced chemical-shift perturbations (CSP) induced in the resonances of the protein and ligand-detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS-NMR). Accuracy, as judged by comparison with X-ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical-shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein-ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein-ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Desenho de Fármacos , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas/metabolismo , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.
