RESUMEN
Peptides representing transmembrane regions of the alpha-subunit of the voltage-gated sodium channel were synthesised and their structures analysed, using 1H NMR and CD, in trifluoroethanol and in dodecylphosphocholine micelles. Sequence analysis suggests that the channel has six regions, S1 to S6, predicted to span the membrane in four homologous domains, designated, I, II, III and IV. Presented here are studies of representatives examples of possible single spanning segments (IS2, IS4, IVS4) and a double spanning segment, IS34, composed of segments IS3 and IS4. In addition, we investigated ISlink56, the putative linker region between segments IS5 and IS6. All of the peptides were found to have predominantly alpha-helical structures in both solvent systems. There was some evidence for bending of the longer helices but there was no discernible evidence for well-defined tertiary structure.
Asunto(s)
Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Canales de Sodio/química , Secuencia de Aminoácidos , Dicroismo Circular , Simulación por Computador , Activación del Canal Iónico , Espectroscopía de Resonancia Magnética , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fosforilcolina/análogos & derivadosRESUMEN
Delta-Toxin is a 26-residue channel-forming peptide from Staphylococcus aureus which forms an amphipathic alpha-helix in a membrane environment. Channel formation in planar bilayers suggests that an average of six delta-toxin helices self-assemble to form transbilayer pores. Molecular models for channels formed by delta-toxin and by a synthetic analogue have been generated using a simulated annealing protocol applied via restrained molecular dynamics. These models are analysed in terms of the predicted geometric and energetic properties of the transbilayer pores. Pore radius calculations of the models demonstrate that rings of channel-lining residues contribute a series of constrictions along the pore. Electrostatic properties of the pores are determined both by pore-lining charged side chains and by the aligned helix dipoles of the parallel helix bundle. Molecular dynamics simulations (100 ps) of delta-toxin models containing intra-pore water were performed. Analysis of the resultant dynamics trajectories further supports the proposal that alternative conformations of pore-constricting side chains may be responsible for the observed conductance heterogeneity of delta-toxin ion channels.
Asunto(s)
Toxinas Bacterianas/química , Canales Iónicos/química , Staphylococcus aureus/química , Secuencia de Aminoácidos , Toxinas Bacterianas/farmacología , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Programas Informáticos , Termodinámica , Agua/química , Agua/metabolismoRESUMEN
We have determined the structure in solution of a homodimeric protein that is a precursor to the locust neuropeptide adipokinetic hormone I using nuclear magnetic resonance spectroscopy. This precursor, called P1, is comprised of two 41 residue strands joined by a single inter-chain disulphide at Cys39. We have also determined the structure of an end product of P1 processing, called APRP1; this is a homodimer comprised of residues 14-41 of P1. Nuclear Overhauser Effect (nOe) data indicate that in both P1 and APRP1, residues 22-37 (numbered with respect to P1) form pairs of alpha-helices, with no evidence for any other secondary structure.
Asunto(s)
Hormonas de Insectos/química , Precursores de Proteínas/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Hormonas de Insectos/biosíntesis , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Precursores de Proteínas/síntesis química , Estructura Secundaria de ProteínaRESUMEN
The neu proto-oncogene may be converted into a dominantly transforming oncogene by a single point mutation. Substitution of a valine residue at position 664 in the transmembrane region with glutamic acid activates the tyrosine kinase of the molecule and is associated with increased receptor dimerization. Previously we have proposed a model in which the glutamic acid side chain stabilizes receptor dimerization by hydrogen bonding. Other models have been proposed in which the mutation leads to a conformational change in the transmembrane region mimicking that assumed to occur following binding of a natural ligand. Synthetic peptides representing part of the transmembrane region were prepared. Some residues were replaced with serine in order to improve peptide solubility to allow purification and analysis. Both the peptides containing valine and glutamic acid dissolved in water and in an artificial lipid monolayer. The structures of the peptides were determined by NMR spectroscopy to be alpha-helical. No significant difference in conformation was observed between the two peptides. This result does not support the model proposing a conformational change. The receptor structures determined experimentally do allow alternative models involving receptor transmembrane region packing.
Asunto(s)
Receptores ErbB/química , Proteínas de la Membrana/química , Proteínas Oncogénicas/química , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Animales , Receptores ErbB/genética , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Oncogénicas/genética , Fragmentos de Péptidos/química , Conformación Proteica , Proteínas Proto-Oncogénicas/genética , Ratas , Receptor ErbB-2RESUMEN
A peptide (S4) of the rat brain sodium channel has been synthesized, studied by high-resolution NMR and its secondary structure modelled by distance geometry and restrained molecular dynamics techniques.
