RESUMO
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.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Mitocôndrias/metabolismo , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/biossíntese , ATPases Translocadoras de Prótons/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Precursores de Proteínas/síntese química , Precursores de Proteínas/química , ATPases Translocadoras de Prótons/síntese química , Espectrofotometria UltravioletaRESUMO
Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.
Assuntos
Membranas Intracelulares/enzimologia , Mitocôndrias/ultraestrutura , ATPases Translocadoras de Prótons/síntese química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Proteínas de Membrana/síntese química , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/ultraestrutura , Leveduras/ultraestruturaRESUMO
A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.