RESUMO
FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Ânions , Sítios de Ligação , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Lipossomos , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Eletricidade EstáticaRESUMO
The signal recognition particle (SRP) of bacteria consists of only one protein, known as Ffh or the SRP54 homologue, which forms a complex with 4.5S RNA. It also binds to signal peptides and contains a GTPase which displays interesting differences to Ras GTPases. The conserved NG-domain of Ffh from the archaebacterium Acidianus ambivalens was cloned and overexpressed with a C-terminal His tag in Escherichia coli. Crystallization experiments of the native protein as well as of the Thr112Ala mutant, which is deficient in GTP hydrolysis, resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 64.5, b = 128.3, c = 72.0 A. At cryogenic temperatures, the crystals diffracted to a resolution limit of 2.8 A using a rotating-anode generator and contain one molecule per asymmetric unit. A native data set has been collected using synchrotron radiation to around 2.0 A resolution. Selenomethionine protein was produced; its crystals diffract in-house to about 2.8 A resolution.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , GTP Fosfo-Hidrolases/química , Partícula de Reconhecimento de Sinal/química , Archaea , Proteínas Arqueais/química , Proteínas de Bactérias/genética , Cristalização , Escherichia coli , Mutação , Fragmentos de Peptídeos/química , Proteínas/química , Proteínas de Ligação a RNA/química , Proteínas Recombinantes/química , Selenometionina/química , Selenoproteínas , Partícula de Reconhecimento de Sinal/genética , Síncrotrons , Difração de Raios XRESUMO
A highly active, large-scale preparation of cytochrome bc1 complex has been obtained from the photosynthetic purple bacterium Rhodovulum (Rhv.) sulfidophilum. It has been characterized using mass spectrometry, quinone and lipid analysis as well as inhibitor binding. About 35 mg of pure complex can be obtained from 1 g of membrane protein. EPR spectroscopy and optical titrations have been used to obtain the redox midpoint potentials of the cofactors. The Em-value of 310 mV for the Rieske protein is the most positive midpoint potential for this protein in a bc1 complex so far. The bc1 complex from Rhv. sulfidophilum is very stable and consists of three subunits and a 6-kDa polypeptide. The complex appears as a dimer in solution and contains six quinone molecules per monomer which are tightly bound. EPR spectroscopy shows that the Q(o) site is highly occupied. High detergent concentrations convert the complex into an inactive, monomeric form that has lost the Rieske protein as well as the quinones and the 6-kDa component.
Assuntos
Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de Elétrons/química , Quinonas/química , Rhodobacter/enzimologia , Sequência de Aminoácidos , Detergentes/farmacologia , Dimerização , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Estabilidade Enzimática , Proteínas Ferro-Enxofre/química , Lipídeos/análise , Espectrometria de Massas , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria UltravioletaRESUMO
Endopeptidase EC 3.4.24.16 (EP24.16c, neurolysin) and thimet oligopeptidase EC 3.4.24.15 are close related members of a large family of metalloproteases. Besides their cytosolic and membrane bound form, endopeptidase EC 3.4.24.16 appears to be present in the inner membrane of the mitochondria (EP24.16m). We have overexpressed two porcine EP24.16 isoforms in E. coli and purified the recombinant proteins to homogeneity. We show here that these peptidases hydrolyse a series of neuropeptides with similar rates and at sites reminiscent of those elicited by classically purified human brain EP24.16c. All neuropeptides, except neurotensin, were similarly cleaved by recombinant endopeptidase 3.4.24.15 (EP24.15, thimet oligopeptidase), another zinc-containing metalloenzyme structurally related to EP24.16. These two EP24.16 isoforms were drastically inhibited by Pro-Ile and dithiothreitol and remained unaffected by a specific carboalkyl inhibitor (CFP-AAY-pAb) directed toward the related EP24.15. The present purification procedure of EP24.16 should allow to establish, by mutagenesis analysis, the mechanistic properties of the enzyme.