RESUMO
Sensory rhodopsin II (NpSRII) is a phototaxis receptor of Natronomonas pharaonis that performs its function in complex with its cognate transducer (NpHtrII). Upon light activation NpSRII triggers by means of NpHtrII a signal transduction chain homologous to the two component system in eubacterial chemotaxis. The D75N mutant of NpSRII, which lacks the blue-shifted M intermediate and therefore exhibits a significantly faster photocycle compared to the wild-type, mediates normal phototaxis responses demonstrating that deprotonation of the Schiff base is not a prerequisite for transducer activation. Using site-directed spin labeling and time resolved electron paramagnetic-resonance spectroscopy, we show that the mechanism revealed for activation of the wild-type complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transmembrane transducer helix TM2, is also valid for the D75N variant. Apparently, the D75N mutation shifts the ground state conformation of NpSRII-D75N and its cognate transducer into the direction of the signaling state.
Assuntos
Substituição de Aminoácidos/genética , Proteínas Arqueais/metabolismo , Carotenoides/metabolismo , Mutação/genética , Natronobacterium/metabolismo , Transdução de Sinais , Proteínas Arqueais/química , Proteínas Arqueais/genética , Carotenoides/química , Carotenoides/genética , Espectroscopia de Ressonância de Spin Eletrônica , Luz , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Natronobacterium/efeitos da radiação , Estrutura Secundária de Proteína , Transdução de Sinais/efeitos da radiação , Marcadores de Spin , Fatores de TempoRESUMO
Colicin A is a water-soluble pore-forming protein that kills cells, which are not protected by an immunity protein, by inserting specific helical segments of the toxin subdomain into the cytoplasmic membrane to form voltage-dependent ion channels. This leads to depolarization of the cell membrane followed by depletion of the intracellular ATP levels and finally to cell death. The formation of the integral membrane voltage-gated ion channel is known to be accompanied by a conformational transition. Using double electron electron resonance spectroscopy inter-spin distances in doubly spin labeled colicin A mutants, with spin labels bound to positions 42/187, 62/187, 91/187 and 115/187, have been determined to serve as constraints for the modeling of the membrane bound, closed channel state of colicin A. The data reveal a quasi-circular arrangement of the eight amphipathic helices, embedded in the membrane interfacial layer close to the lipid-water interface, whereas the two hydrophobic helices are buried within the membrane.
Assuntos
Colicinas/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli/química , Bicamadas Lipídicas/química , Substituição de Aminoácidos/genética , Colicinas/genética , Escherichia coli/genética , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Marcadores de Spin , Lipossomas Unilamelares/químicaRESUMO
HAMP domains (conserved in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) perform their putative function as signal transducing units in diversified environments in a variety of protein families. Here the conformational changes induced by environmental agents, namely salt and temperature, on the structure and function of a HAMP domain of the phototransducer from Natronomonas pharaonis (NpHtrII) in complex with sensory rhodopsin II (NpSRII) were investigated by site-directed spin labeling electron paramagnetic resonance. A series of spin labeled mutants were engineered in NpHtrII157, a truncated analog containing only the first HAMP domain following the transmembrane helix 2. This truncated transducer is shown to be a valid model system for a signal transduction domain anchored to the transmembrane light sensor NpSRII. The HAMP domain is found to be engaged in a "two-state" equilibrium between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation. The structural properties of the cHAMP as proven by mobility, accessibility, and intra-transducer-dimer distance data are in agreement with the four helical bundle NMR model of the HAMP domain from Archaeoglobus fulgidus.
Assuntos
Halobacteriaceae/metabolismo , Sais/farmacologia , Sequência de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica , Halorrodopsinas/química , Luz , Transdução de Sinal Luminoso , Espectroscopia de Ressonância Magnética , Conformação Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Rodopsinas Sensoriais/química , Transdução de SinaisRESUMO
The nature and kinetics of the conformational changes leading to the activated state of NpSRII/NpHtrII157 were investigated by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy in combination with site-directed spin labeling (SDSL) on a series of spin labeled mutants of NpSRII. A structural rearrangement of the cytoplasmic moiety of NpSRII upon light activation was detected (helices B, C, F and G). The increase in distance between helices C and F in the M-trapped state of the complex observed in one double mutant is in line with the notion that an outward movement of helix F occurs upon receptor activation. The data obtained from the NpSRII/NpHtrII157 complex reconstituted in purple membrane lipids are compared with those obtained from the X-ray structure of the late M-state of the complex which shows some discrepancies. The results are discussed in the context also of other biophysical and EPR experimental evidences.
