RESUMO
A new strategy was established to determine the average orientation and dynamics of ergosterol in dimyristoylphosphatidylcholine model membranes. It is based on the analysis of chemical shift anisotropies (CSAs) averaged by the molecular dynamics. Static (13)C CSA tensors were computed by quantum chemistry, using the gauge-including atomic-orbital approach within Hartree-Fock theory. Uniformly (13)C-labeled ergosterol was purified from Pichia pastoris cells grown on labeled methanol. After reconstitution into dimyristoylphosphatidylcholine lipids, the complete (1)H and (13)C assignment of ergosterol's resonances was performed using a combination of magic-angle spinning two-dimensional experiments. Dynamically averaged CSAs were determined by standard side-band intensity analysis for isolated (13)C resonances (C(3) and ethylenic carbons) and by off-magic-angle spinning experiments for other carbons. A set of 18 constraints was thus obtained, from which the sterol's molecular order parameter and average orientation could be precisely defined. The validity of using computed CSAs in this strategy was verified on cholesterol model systems. This new method allowed us to quantify ergosterol's dynamics at three molar ratios: 16 mol % (Ld phase), 30 mol % (Lo phase), and 23 mol % (mixed phases). Contrary to cholesterol, ergosterol's molecular diffusion axis makes an important angle (14 degrees) with the inertial axis of the rigid four-ring system.
Assuntos
Dimiristoilfosfatidilcolina/química , Ergosterol/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Fluidez de Membrana , Modelos Químicos , Modelos Moleculares , Anisotropia , Isótopos de Carbono , Simulação por Computador , Difusão , Ergosterol/análise , Cinética , Membranas Artificiais , Pichia/metabolismo , Conformação ProteicaRESUMO
Molecular segregation in methanol-water mixtures is studied across a wide concentration range as a function of temperature and pressure. Cluster distributions obtained from both neutron diffraction and molecular dynamics simulations point to significantly enhanced segregation as the mixtures are cooled or compressed. This evolution toward greater molecular heterogenity in the mixture accounts for the observed changes in the water-water radial distribution function and there are indications also of a change in the topology of the water clusters. The observed behavior is consistent with an approach to an upper critical solution point. Such a point would appear to be "hidden" below the freezing line, thereby precluding observation of the two-fluid region.
RESUMO
The hydration and dynamics of purple membranes (PM) containing the bacteriorhodopsin (BR) triple mutant D96G/F171C/F219L were investigated by neutron diffraction coupled with H(2)O/D(2)O exchange and by energy-resolved neutron scattering. The mutant, which is active in proton transport (Tittor et al. in J. Mol. Biol. 319:555-565, 2002), has an "open" ground-state structure similar to that of the M intermediate in the photocycle of the wild type (wt) (Subramaniam and Henderson in Nature 406:653-657, 2000). The experiments demonstrated an increased proton channel hydration in the mutant PM compared with wt PM, in both high (86%) and low (57%) relative humidity. We suggest that this is due to the smaller side chains of the mutant residues liberating space for more water molecules in the proton channel, which would then be able to participate in the proton translocation network. PM thermal dynamics has been shown to be very sensitive to membrane hydration (Lehnert et al. in Biophys. J. 75:1945-1952, 1998). The global dynamical behaviour of the mutant PM on the 100-ps time scale, as a function of relative humidity, was found to be identical to that of the wt, showing that the "open" BR structure and additional water molecules in the proton channel do not provide a softer environment enabling increased flexibility.
Assuntos
Bacteriorodopsinas/genética , Mutação , Prótons , Membrana Celular/metabolismo , Óxido de Deutério/química , Halobacterium salinarum/metabolismo , Temperatura Alta , Luz , Lipídeos/química , Conformação Molecular , Nêutrons , Conformação Proteica , Membrana Purpúrea/metabolismo , Espalhamento de Radiação , Temperatura , Água/químicaRESUMO
An extensive series of neutron diffraction experiments and molecular dynamics simulations has shown that mixtures of methanol and water exhibit extended structures in solution despite the components being fully miscible in all proportions. Of particular interest is a concentration region (methanol mole fraction between 0.27 and 0.54) where both methanol and water appear to form separate, percolating networks. This is the concentration range where many transport properties and thermodynamic excess functions reach extremal values. The observed concentration dependence of several of these material properties of the solution may therefore have a structural origin.
RESUMO
The quality and signal to noise ratio of a J-based HETCOR performed on a standard MAS probe have been compared with a gradient enhanced HSQC performed on a HR-MAS probe at 500 MHz. The sample selected was cholesterol, inserted at 30 mol% in acyl chain deuterated phospholipids (DMPC-d54), at a temperature where the bilayer is in a liquid crystalline phase (310 K). It is representative of any rigid molecule undergoing fast axial diffusion in a bilayer as the main movement. After optimization of the spinning rate and carbon decoupling conditions, it is shown that the ge-HSQC/MAS approach is far superior to the more conventional J-HETCOR/MAS in terms of signal to noise ratio, and that it allows the detection of all the natural abundance cross peaks of cholesterol in a membrane environment. Clear differences between the 1H and 13C chemical shifts of cholesterol in a membrane and in chloroform solution were thus revealed.
Assuntos
Colesterol/química , Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Fluidez de Membrana , Ressonância Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono , Colesterol/análise , Dimiristoilfosfatidilcolina/análise , Estudos de Viabilidade , Bicamadas Lipídicas/análise , Lipídeos de Membrana/análise , Lipídeos de Membrana/química , Prótons , Marcadores de Spin/síntese químicaRESUMO
13C NMR spectra routinely performed on oriented lipid bilayers display linewidth of 1-2 ppm, although T2 measurements indicate that 0.1-0.2 ppm could be obtained. We have prepared a DMPC-13C4-cholesterol (7/3) sample, and oriented the lipid bilayers between glass plates so that the bilayer normal makes an angle of 90 degrees (or of the magic angle) with Bo. We have measured T2s, CSAs, and linewidths for the choline 13C-gamma-methyl, the cholesterol-C4 carbons and the lipid head group phosphorus, at both angles and 313 K. The magnetic field distribution within the sample was calculated using the surface current formalism. The line shapes were simulated as a function of Bo field inhomogeneities and sample mosaic spread. Both effects contribute to the experimental linewidth. Using three signals of different CSA, we have quantified both contributions and measured the mosaic spread accurately. Direct shimming on a sample signal is essential to obtain sharp resonances and 13C labelled choline methyl resonance of DMPC is a good candidate for this task. After optimisation of the important parameters (shimming on the choline resonance, mosaic spread of +/-0.30 degrees), 13C linewidth of 0.2-0.3 ppm have been obtained. This newly achieved resolution on bilayers oriented at 90 degrees, has allowed to perform two 2D experiments, with a good sensitivity: 2D PELF (correlation of carbon chemical shifts and C-H dipolar couplings) and 2D D-resolved experiment (correlation of carbon chemical shifts and C-C dipolar couplings). A C-C dipolar coupling of 35 +/- 2 Hz between the choline methyl carbons was determined.
Assuntos
Carbono/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Anisotropia , Colesterol/análogos & derivados , Colesterol/química , Colina/análogos & derivados , Colina/química , Dimiristoilfosfatidilcolina/análogos & derivados , Dimiristoilfosfatidilcolina/química , Lipossomos/química , Conformação MolecularRESUMO
A transition as a function of increasing temperature from harmonic to anharmonic dynamics has been observed in globular proteins by using spectroscopic, scattering, and computer simulation techniques. We present here results of a dynamic neutron scattering analysis of the solvent dependence of the picosecond-time scale dynamic transition behavior of solutions of a simple single-subunit enzyme, xylanase. The protein is examined in powder form, in D(2)O, and in four two-component perdeuterated single-phase cryosolvents in which it is active and stable. The scattering profiles of the mixed solvent systems in the absence of protein are also determined. The general features of the dynamic transition behavior of the protein solutions follow those of the solvents. The dynamic transition in all of the mixed cryosolvent-protein systems is much more gradual than in pure D(2)O, consistent with a distribution of energy barriers. The differences between the dynamic behaviors of the various cryosolvent protein solutions themselves are remarkably small. The results are consistent with a picture in which the picosecond-time scale atomic dynamics respond strongly to melting of pure water solvent but are relatively invariant in cryosolvents of differing compositions and melting points.
Assuntos
Proteínas/química , Xilosidases/química , Varredura Diferencial de Calorimetria , Simulação por Computador , Óxido de Deutério , Congelamento , Nêutrons , Espalhamento de Radiação , Soluções , Solventes , Termodinâmica , Difração de Raios X , Xilano Endo-1,3-beta-XilosidaseRESUMO
The activity and dynamics of a simple, single subunit enzyme, the xylanase from Thermotoga maritima strain Fj SS3B.1 have been measured under similar conditions, from -70 to +10 degrees C. The internal motions of the enzyme, as evidenced by neutron scattering, undergo a sharp transition within this temperature range; they show no evidence for picosecond-timescale anharmonic behaviour (e.g. local diffusive motions or jumps between alternative conformations) at temperatures below -50 degrees C, whereas these motions are strongly activated at higher temperatures. The activity follows Arrhenius behaviour over the whole of the temperature range investigated, -70 to +10 degrees C. The results indicate that a temperature range exists over which the enzyme rate-limiting step is independent of fast anharmonic dynamics.
Assuntos
Xilosidases/química , Ativação Enzimática , Escherichia coli , Temperatura , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/metabolismoRESUMO
For the study of protein structure, dynamics, and function, at very low temperatures it is desirable to use cryosolvents that resist phase separation and crystallisation. We have examined these properties in a variety of cryosolvents. Using visual and X-ray diffraction criteria, methanol:ethanediol (70%:10%), methanol:glycerol (70%:10%), acetone:methoxy-ethanol:ethanediol (35%:35%:10%), dimethylformamide:ethanediol (70%:10%), dimethylformamide (80%), methoxyethanol (80%), and methoxyethanol:ethanediol (70%:10%) were all found to be free of phase-changes down to at least -160 degrees C. The least viscous of these, methanol:ethanediol (70%:10%), was miscible down to -125 degrees C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology.
Assuntos
Crioprotetores , Enzimas/química , Enzimas/metabolismo , Proteínas/química , Solventes , Proteínas/metabolismoRESUMO
We have examined the temperature dependence of motions in a cryosolution of the enzyme glutamate dehydrogenase (GDH) and compared these with activity. Dynamic neutron scattering was performed with two instruments of different energy resolution, permitting the separate determination of the average dynamical mean square displacements on the sub-approximately 100 ps and sub-approximately 5 ns time scales. The results demonstrate a marked dependence on the time scale of the temperature profile of the mean square displacement. The lowest temperature at which anharmonic motion is observed is heavily dependent on the time window of the instrument used to observe the dynamics. Several dynamical transitions (inflexions of the mean squared displacement) are observed in the slower dynamics. Comparison with the temperature profile of the activity of the enzyme in the same solvent reveals dynamical transitions that have no effect on GDH function.
Assuntos
Glutamato Desidrogenase/metabolismo , Thermococcus/enzimologia , Varredura Diferencial de Calorimetria , Fenômenos Químicos , Físico-Química , Cristalização , Cristalografia por Raios X , Elasticidade , Glutamato Desidrogenase/química , Cinética , Nêutrons , Relação Estrutura-Atividade , Temperatura , Termodinâmica , Fatores de TempoRESUMO
Bacteriorhodopsin (BR) is a transmembrane protein in the purple membrane (PM) of Halobacterium salinarum. Its function as a light-driven proton pump is associated with a cycle of photointermediates which is strongly hydration-dependent. Using energy-resolved neutron scattering, we analyzed the thermal motions (in the nanosecond-to-picosecond time range) in PM at different hydration levels. Two main populations of motions were found that responded differently to water binding. Striking correlations appeared between these "fast" motions and the "slower" kinetic constants (in the millisecond time range) of relaxations and conformational changes occurring during the photocycle.
Assuntos
Bacteriorodopsinas/química , Conformação Proteica/efeitos da radiação , Bacteriorodopsinas/efeitos da radiação , Elasticidade , Halobacterium salinarum , Umidade , Cinética , Luz , Nêutrons , Distribuição Normal , Espalhamento de Radiação , Temperatura , TermodinâmicaRESUMO
We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing H-retinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.