RESUMEN
The 16-amino acid sequences of the C-terminal helices of the homologous bacterial cytochromes c551 from Pseudomonas aeruginosa and C552 from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of alpha-helices and 3(10)-helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c551 and 20% for c552, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2-trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2-trifluoroethanol. NMR spectra in phosphate buffer show the presence of both alpha- and 3(10)-helical structures. In water/2,2,2-trifluoroethanol (50:50) alpha-helices are predominant. CD temperature-dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2-trifluoroethanol (50:50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c552 C-terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.
Asunto(s)
Bacterias/química , Proteínas Bacterianas , Dicroismo Circular , Grupo Citocromo c/química , Espectroscopía de Resonancia Magnética , Pseudomonas aeruginosa/química , Algoritmos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Temperatura , Trifluoroetanol/químicaAsunto(s)
Aminoácido Oxidorreductasas/química , Proteínas Portadoras/química , Isoenzimas/química , Resonancia Magnética Nuclear Biomolecular , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas , Proteínas Portadoras/metabolismo , Bases de Datos Factuales , Escherichia coli/química , Complejo Glicina-Descarboxilasa , Proteína H del Complejo de la Glicina Descarboxilasa , Glicina-Deshidrogenasa (Descarboxilante) , Metilaminas/metabolismo , Oxidación-ReducciónRESUMEN
The 26-amino-acid pre-sequence of the ATP synthase beta subunit that directs the protein from the cytosol to mitochondria in the unicellular green alga Chlamydomonas reinhardtii has been synthesised and analysed using NMR spectroscopy/circular dichroism and compared to a chloroplast transit peptide from the same organism. The results demonstrate that the peptide, though mainly unstructured in water, undergoes a strong conformational change in a 36% water/64% 2,2,2-trifluoroethanol mixture. In this solvent condition, an alpha-helix was characterised by NMR from residue 2 to 26. Structure calculations under NMR restraints lead to a population of models of which 60% are kinked at position 9-10. Structural analysis indicates two hydrophobic sectors on the models with a discontinuity at the 9-10 kink level. The structures suggest a different interaction mode with the mitochondrial membrane compared to the chloroplast transit peptide.