RESUMO
The solution structure of the DNA-binding domain of the TraM protein, an essential component of the DNA transfer machinery of the conjugative resistance plasmid R1, is presented. The structure has been determined using homonuclear 2-dimensional NMR spectroscopy as well as 15N labeled heteronuclear 2- and 3-dimensional NMR spectroscopy. It turns out that the solution structure of the DNA binding domain of the TraM protein is globular and dominantly helical. The very first amino acids of the N-terminus are unstructured.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Plasmídeos/química , Conformação Proteica , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Soluções , TermodinâmicaRESUMO
To test the hypothesis that the folding pathways of evolutionarily related proteins with similar three-dimensional structures but widely different sequences should be similar, the folding pathway of apoleghemoglobin has been characterized using stopped-flow circular dichroism, heteronuclear NMR pulse labeling techniques and mass spectrometry. The pathway of folding was found to differ significantly from that of a protein of the same family, apomyoglobin, although both proteins appear to fold through helical burst phase intermediates. For leghemoglobin, the burst phase intermediate exhibits stable helical structure in the G and H helices, together with a small region in the center of the E helix. The A and B helices are not stabilized until later stages of the folding process. The structure of the burst phase folding intermediate thus differs from that of apomyoglobin, in which stable helical structure is formed in the A, B, G and H helix regions.
Assuntos
Evolução Molecular , Globinas/química , Globinas/metabolismo , Hemeproteínas/química , Hemeproteínas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Cinética , Leghemoglobina/química , Leghemoglobina/metabolismo , Espectrometria de Massas , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mioglobina/química , Mioglobina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica/efeitos dos fármacos , Renaturação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Termodinâmica , Ureia/farmacologiaRESUMO
A synthetic gene for apoleghemoglobin-a from soybean, optimized for expression in Escherichia coli has been designed and synthesized by a recursive polymerase chain reaction technique. The protein has been expressed with high efficiency and a purification protocol has been developed. The holoprotein is readily reconstituted by the addition of heme. 15N- and 15N,13C-labeled samples were produced and backbone 15N and 13C assignments were determined by 2D and 3D NMR spectroscopy. Comparison of the chemical shifts of 13C(alpha) and 13CO with random coil shifts revealed a pattern of secondary structure which correlates well with the one previously derived from homonuclear NMR data and low-resolution X-ray crystallography.
Assuntos
Expressão Gênica , Glycine max/química , Leghemoglobina/genética , Sequência de Aminoácidos , Sequência de Bases , Isótopos de Carbono , Clonagem Molecular , DNA Recombinante , Escherichia coli/genética , Leghemoglobina/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Isótopos de NitrogênioRESUMO
Complete resonance assignments for the 13C spectrum of reduced (Cu(I)) rusticyanin have been made using 13C, 15N doubly labeled recombinant material. The reported assignments include those for the carboxyl and carbonyl carbon atoms and protonated aromatic ring carbons, and were obtained using a variety of 2- and 3D inverse-detected NMR experiments, including 13C, 15N, 1H triple resonance experiments and HCCH-COSY and -TOCSY. Backbone carbonyl assignments were obtained using 3D HNCO and HCACO spectra, and modified versions of 2D H(CA)CO and HMBC spectra were used to obtain side-chain carboxyl carbon and methionine epsilon-methyl carbon assignments, respectively. A comparison of the 13C alpha, 13C beta and 13CO chemical shifts with published 'random coil' values confirms the conclusion reached from a consideration of the 3JHN alpha coupling constants and the pattern of sequential NOEs, that the protein consists largely of beta-structure.
Assuntos
Azurina/análogos & derivados , Proteínas de Bactérias/química , Cobre/química , Estrutura Secundária de Proteína , Thiobacillus/química , Sequência de Aminoácidos , Azurina/química , Azurina/genética , Isótopos de Carbono , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oxirredução , Prolina/química , Prótons , Proteínas Recombinantes/químicaRESUMO
The major light-harvesting complex (LHCII) of photosystem II, the most abundant chlorophyll-containing complex in higher plants, is organized in trimers. In this paper we show that the trimerization of LHCII occurs spontaneously and is dependent on the presence of lipids. LHCII monomers were reconstituted from the purified apoprotein (LHCP), overexpressed in Escherichia coli, and pigments, purified from chloroplast membranes. These synthetic LHCII monomers trimerize in vitro in the presence of a lipid fraction isolated from pea thylakoids. The reconstituted LHCII trimers are very similar to native LHCII trimers in that they are stable in the presence of mild detergents and can be isolated by partially denaturing gel electrophoresis or by centrifugation in sucrose density gradients. Moreover, both native and reconstituted LHCII trimers exhibit signals in circular dichroism in the visible range that are not seen in native or reconstituted LHCII monomers, indicating that trimer formation either establishes additional pigment-pigment interactions or alters pre-existing interactions. Reconstituted LHCII trimers readily form two-dimensional crystals that appear to be identical to crystals of the native complex.
