RESUMO
Small heat shock proteins (sHsps) play a role in preventing the fatal aggregation of denatured proteins in the presence of stresses. The sHsps exist as monodisperse oligomers in their resting state. Because the hydrophobic N-terminal regions of sHsps are possible interaction sites for denatured proteins, the manner of assembly of the oligomer is critical for the activation and inactivation mechanisms. Here, we report the oligomer architecture of SpHsp16.0 from Schizosaccharomyces pombe determined with X-ray crystallography and small angle X-ray scattering. Both results indicate that eight dimers of SpHsp16.0 form an elongated sphere with 422 symmetry. The monomers show nonequivalence in the interaction with neighboring monomers and conformations of the N- and C-terminal regions. Variants for the N-terminal phenylalanine residues indicate that the oligomer formation ability is highly correlated with chaperone activity. Structural and biophysical results are discussed in terms of their possible relevance to the activation mechanism of SpHsp16.0.
Assuntos
Proteínas de Choque Térmico/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Proteínas de Choque Térmico/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Proteínas de Schizosaccharomyces pombe/genética , Soluções , Homologia Estrutural de ProteínaRESUMO
Small heat-shock proteins (sHsps) bind and stabilize proteins denatured by heat or other stresses in order to prevent unfavourable protein aggregation. StHsp14.0 is an sHsp found in the acidothermophilic archaeon Sulfolobus tokodaii. A variant of StHsp14.0 was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted X-rays to 1.85 A resolution and belonged to space group P2(1)2(1)2, with unit-cell parameters a = 40.4, b = 61.1, c = 96.1 A. The V(M) value was estimated to be 2.1 A(3) Da(-1), assuming the presence of two molecules in the asymmetric unit. Heavy-atom derivative crystals were prepared successfully by the cocrystallization method and are isomorphic to native crystals.
Assuntos
Proteínas de Choque Térmico Pequenas/química , Sulfolobus/química , Brometos/química , Cloretos/química , Cobalto/química , Cristalização , Cristalografia por Raios X , Proteínas de Choque Térmico Pequenas/metabolismo , Compostos de Manganês/química , Rubídio/química , Compostos de Sódio/química , Estrôncio/químicaRESUMO
Lactate is utilized in many biological processes, and its transport across biological membranes is mediated with various types of transporters. Here, we report the crystal structures of a lactate-binding protein of a TRAP (tripartite ATP-independent periplasmic) secondary transporter from Thermus thermophilus HB8. The folding of the protein is typical for a type II periplasmic solute-binding protein and forms a dimer in a back-to-back manner. One molecule of l-lactate is clearly identified in a cleft of the protein as a complex with a calcium ion. Detailed crystallographic and biochemical analyses revealed that the calcium ion can be removed from the protein and replaced with other divalent cations. This characterization of the structure of a protein binding with calcium lactate makes a significant contribution to our understanding of the mechanisms by which calcium and lactate are accommodated in cells.
Assuntos
Compostos de Cálcio/química , Lactatos/química , Proteínas de Membrana Transportadoras/química , Proteínas Periplásmicas de Ligação/química , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Compostos de Cálcio/metabolismo , Cristalografia por Raios X , Lactatos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Dobramento de Proteína , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Thermus thermophilus/químicaRESUMO
From June 2000 to September 2001, we investigated the presence of eggs spawned in Margaritifera laevis and the seasonal changes in the gonads of Tanakia tanago. Eggs were observed from mid-March to mid-September. In females with a shrunken ovipositor, as the GSI gradually increased, most ovaries were in the prespawning phase (Oct-Mar). As the GSI increased further, most ovaries were in the early spawning phase (Mar-Jun). As the GSI gradually deceased, ovaries in the late spawning phase appeared (Jun-Sep). When the GSI was very low, most ovaries were in the postspawning phase (Sep-Oct). In males, when the GSI was low, most testes were in the early prespawning phase from Oct-Dec. As the GSI gradually increased, most testes were in the late prespawning phase (Dec-Jan). As the GSI increased further, testes were in the early spawning phase (Jan-Jun). As the GSI gradually decreased, amost testes were in the late spawning phase (Jun-Sep). When the GSI was very low, most testes were in the postspawning phase (Sep-Oct). These results indicate that T. tanago has a distinct annual reproductive cycle and is a spring-autumn spawner. Based on the relationship between reproductive activity and environmental factors, the spawning season of T. tanago appears to be initiated by increasing temperature and / or longer days in spring and to be terminated by shorter days in autumn.
