共查询到20条相似文献,搜索用时 31 毫秒
1.
Anna Diller Cécile Loudet Fabien Aussenac Gérard Raffard Sylvie Fournier Michel Laguerre Axelle Grélard Stanley J. Opella Francesca M. Marassi Erick J. Dufourc 《Biochimie》2009
Major biological processes occur at the biological membrane. One of the great challenges is to understand the function of chemical or biological molecules inside the membrane; as well of those involved in membrane trafficking. This requires obtaining a complete picture of the in situ structure and dynamics as well as the topology and orientation of these molecules in the membrane lipid bilayer. These led to the creation of several innovative models of biological membranes in order to investigate the structure and dynamics of amphiphilic molecules, as well as integral membrane proteins having single or multiple transmembrane segments. Because the determination of the structure, dynamics and topology of molecules in membranes requires a macroscopic alignment of the system, a new membrane model called ‘bicelles’ that represents a crossover between lipid vesicles and classical micelles has become very popular due to its property of spontaneous self-orientation in magnetic fields. In addition, crucial factors involved in mimicking natural membranes, such as sample hydration, pH and salinity limits, are easy to control in bicelle systems. Bicelles are composed of mixtures of long chain (14–18 carbons) and short chain phospholipids (6–8 carbons) hydrated up to 98% with buffers and may adopt various morphologies depending on lipid composition, temperature and hydration. We have been developing bicelle systems under the form of nano-discs made of lipids with saturated or biphenyl-containing fatty acyl chains. Depending on the lipid nature, these membranous nano-discs may be macroscopically oriented with their normal perpendicular or parallel to the magnetic field, providing a natural ‘molecular goniometer’ for structural and topological studies, especially in the field of NMR. Bicelles can also be spun at the magic angle and lead to the 3D structural determination of molecules in membranes. 相似文献
2.
Loudet C Khemtémourian L Aussenac F Gineste S Achard MF Dufourc EJ 《Biochimica et biophysica acta》2005,1724(3):315-323
Mixtures of dicaproyl- (DC), dimyristoyl- (DM) and 1-tetradecanoyl-2-biphenylbutanoyl-(TBB) phosphatidylcholine (PC) in water produce bicelle membranes that are oriented by magnetic fields. DMPC/DCPC systems orient such that their membrane plane is parallel to the magnetic field, whereas for TBBPC/DCPC, the plane is perpendicular to the field. Partial temperature-composition-hydration diagrams are established using solid-state 31P-NMR. DMPC/DCPC bicelles exist on a large range of composition but on a narrow temperature domain (25-45 degrees C). At converse, TBBPC/DCPC form bicelles on a narrow compositional range but over a large temperature span (10-70 degrees C). The TBBPC/DCPC bicelles are shown to be a very powerful potential tool to study the orientation of hydrophobic helices in membranes using wide line 15N-NMR. The DMPC/DCPC system that undergoes a micelle-to-bicelle transition on going from 10 degrees C to 40 degrees C may be used with circular dichroism to study the state of association of hydrophobic helices within the membrane. Results suggest that the transmembrane fragment of the neu/erbB-2 receptor is monomeric in micellar medium and dimeric/multimeric in bicelle membranes. 相似文献
3.
Luchette PA Vetman TN Prosser RS Hancock RE Nieh MP Glinka CJ Krueger S Katsaras J 《Biochimica et biophysica acta》2001,1513(2):83-94
Bilayered micelles, or bicelles, which consist of a mixture of long- and short-chain phospholipids, are a popular model membrane system. Depending on composition, concentration, and temperature, bicelle mixtures may adopt an isotropic phase or form an aligned phase in magnetic fields. Well-resolved (1)H NMR spectra are observed in the isotropic or so-called fast-tumbling bicelle phase, over the range of temperatures investigated (10-40 degrees C), for molar ratios of long-chain lipid to short-chain lipid between 0.20 and 1.0. Small angle neutron scattering data of this phase are consistent with the model in which bicelles were proposed to be disk-shaped. The experimentally determined dimensions are roughly consistent with the predictions of R.R. Vold and R.S. Prosser (J. Magn. Reson. B 113 (1996)). Differential paramagnetic shifts of head group resonances of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC), induced by the addition of Eu(3+), are also consistent with the bicelle model in which DHPC is believed to be primarily sequestered to bicelle rims. Selective irradiation of the DHPC aliphatic methyl resonances results in no detectable magnetization transfer to the corresponding DMPC methyl resonances (and vice versa) in bicelles, which also suggests that DHPC and DMPC are largely sequestered in the bicelle. Finally, (1)H spectra of the antibacterial peptide indolicidin (ILPWKWPWWPWRR-NH(2)) are compared, in a DPC micellar phase and the above fast-tumbling bicellar phases for a variety of compositions. The spectra exhibit adequate resolution and improved dispersion of amide and aromatic resonances in certain bicelle mixtures. 相似文献
4.
An apparatus is described which is designed for preparative freeze concentration experiments by the technique of normal freezing. It has a capacity of approximately 22 liters distributed over twelve vessels. The influence of various geometrical and chemical parameters such as stirring speed, crystallization rate, and sample composition on the normal freezing of protein solutions are discussed. For dilute protein solutions (<0.1%) the concentration factor generally was 8- to 10-fold with recoveries of 90–100 percent. With higher protein concentrations and at ionic strengths higher than approximately 0.05, the recovery was decreased. No loss of activity was detected when concentrating enzyme solutions. 相似文献
5.
Virgen-Ortíz JJ Ibarra-Junquera V Osuna-Castro JA Escalante-Minakata P Mancilla-Margalli NA Ornelas-Paz Jde J 《Analytical biochemistry》2012,426(1):4-12
One of the recurrent methodological problems in preparative biochemical work is the concentration of dilute protein solutions, including culture supernatants resulting from biotechnological processes. A procedure was developed to concentrate enzymes by a novel cryoconcentration system. This approach includes a new device that facilitates the sample freezing and the subsequent solute elution from the frozen matrix by centrifugation. The optimal centrifugation conditions for this cryoconcentration system were obtained using whey protein solution as a model. The procedure was applied to concentrate dilute solutions of commercial pectinase, measuring the endopolygalacturonase (EPG) activity of this enzyme in the concentrate by a method based on the on-line torque measurement, and of recombinant fructan:fructan 1-fructosyltransferase (1-FFT) protein of Pichia pastoris from a culture in a bioreactor, as an expression system. The optimal centrifugation speed, time, and temperature were 6150 g, 20 min, and 4 °C, respectively. The concentration factors for the dilute protein solutions were 9.2-, 11.2-, and 17.1-fold for 1-FFT, whey, and commercial pectinase, respectively. Recoveries ranged from 87% to 93%. The procedure allowed concentrating proteins efficiently without affecting their enzymatic activity. 相似文献
6.
We describe the capillary flow behavior of gels of beta-lactoglobulin (beta-lg) containing droplets of fibrils and the shear flow alignment of beta-lg fibers in dilute aqueous solutions. Polarized optical microscopy and laser scanning confocal microscopy are used to show that capillary shear flow does not affect the fibril droplet sizes in the beta-lg gels, the system behaving in this respect as a solution of compact colloidal particles under shear flow. Small-angle X-ray scattering (SAXS) on dilute aqueous solutions indicates that the fibers can be initially aligned under capillary shear, but this alignment is lost after 18 min of shear. Transmission electron microscopy experiments on the samples studied by SAXS suggest that the loss of orientation is due to a shear-induced breakup of the swollen fibril network. Dynamic and static light scattering on dilute beta-lg fibril aqueous solutions are used to show that before shear beta-lg fibrils behave as strongly interacting semiflexible polymers, while they behave as weakly interacting rods after 18 min of capillary shear. 相似文献
7.
Characterization of proteoglycan and the proteoglycan--hyaluronic acid complex by electric birefringence. 总被引:1,自引:0,他引:1
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An electric field causes partial alignment of macromolecules in a dilute solution. The accompanying changes in the solution birefringence offer a sensitive and quick means of monitoring the rates of particle orientation and hence the size of the solute molecules. Such measurements are reported for dilute solutions of proteoglycans in the absence and presence of added hyaluronic acid. The proteoglycan molecules are shown to be some 580 nm long. In the presence of hyaluronic acid they form aggregates that appear to be consistent with the model previously proposed in which the proteoglycans attach radially to the extended hyaluronic acid chain. The electric-birefringence relaxation rates indicate aggregates of similar length to that of the extended hyaluronic acid chain, with the proteoglycans spaced on average at 29nm intervals. A proteoglycan sample the cystine residues of which had been reduced and alkylated showed no evidence of aggregation with hyaluronic acid up to the concentrations of the acid corresponding to 1% of the total uronic acid content. The electric-birefringence method is shown to have a large potential in the study of associating polysaccharide solutions. 相似文献
8.
Cation modulation of bicelle size and magnetic alignment as revealed by solid-state NMR and electron microscopy
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The influence of salts (KCl, NaCl, CaCl(2), and MgCl(2)) on bicelles (bilayered micelles) made of dimyristoylphosphatidylcholine (DMPC, molar fraction X = 78%) and dicaproylphosphatidylcholine (DCPC) was investigated by solid-state (31)P- and (2)H NMR as well as by freeze-fracture electron microscopy. Sizes were determined from (2)H- and (31)P NMR on the basis of a model that incorporated a planar bilayer and a (half-torus) curved rim representing the DMPC and DCPC regions of the bicelle, respectively. Good agreement was shown with sizes determined independently from freeze-fracture electron microscopy images. In the presence of K(+) and Na(+), bicelles have diameters of approximately 300 A while in the presence of Ca(2+) and Mg(2+); their diameter increases to approximately 500 A. Bicelle magnetic alignment is considerably improved by the presence of salts. The optimum salt concentration for such an effect ranges from 50 to 200 mM. Bicelles are magnetically aligned for temperatures roughly ranging from 30 degrees C to 40 degrees C with monovalent cations; this range is slightly extended in the presence of divalent salts. In this temperature range, the dynamics of the long-chain hydrocarbon region of the bicelle (leading to a bicelle thickness of 38 A) and of water is about the same independently of cation nature and concentration. However, at higher temperatures, considerable differences in water dynamics are observed between systems with monovalent and divalent cations. In these conditions, the system consists of a mixture of micelles and extended bilayers, which show residual macroscopic alignment in the magnetic field. 相似文献
9.
Marise Ouellet 《生物化学与生物物理学报:生物膜》2010,1798(2):235-1094
We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, 31P and 2H NMR experiments were performed on 21-mer peptide-containing bicelles. 31P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, 2H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. 15N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. 15N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, 13C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the 13CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation. 相似文献
10.
Low Temperature Absorption Spectra of Chlorophyll a in Polar and Nonpolar Solvents 总被引:1,自引:1,他引:0
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Absorption spectra of chlorophyll a were measured in polar and non-polar solvents, as a function of temperature from 298 degrees to 77 degrees K. Both dilute and concentrated solutions were examined. In both types of solvents at room temperature, the absorption spectra of concentrated solutions differ from dilute ones in that the half width of the main red absorption band is greater, and all bands are shifted to longer wavelengths. These differences are largely due to the presence of dimers when the pigment concentration is high. In dilute ethanol solutions, where the chlorophyll is unassociated, cooling causes a red shift in all bands which is due to the increased polarity of the solvent at low temperature. On cooling at high concentrations in ethanol and EPA, a new band appears near 700 nm. This band is attributed to dimers present prior to cooling, but absorbing at shorter wavelengths at room temperature. In nonpolar solvents, a band near 700 nm appears at the solvent freezing point. In these solvents, the "700" nm absorption is attributed to dimers, and/or small polymers, partly formed by cooling. A change in aggregate geometry when the solvent becomes viscous or frozen can account for the appearance of this "700" nm absorption band at low temperature, in polar and nonpolar media. 相似文献
11.
The stability and viability of Neisseria gonorrhoeae WP (T4) was tested in a variety of buffers and solutions, many of which are commonly used in gonococcal research. Each solution was tested at room temperature for its ability to maintain stability and viability of gonococci in concentrated suspensions and to maintain viability of gonococci in dilute suspensions. The 14 buffers and solutions tested could be divided into four groups based upon these criteria. Only a few solutions satisfied all three criteria. Of those tested, Gey salt solution and bovine serum albumin (0.01%) and proteose peptone (1%) in saline were the only two in which the gonococci retained viability in dilute suspensions for 25 min. Most of the solutions were not able to maintain viability of gonococci in dilute suspensions, even when the same solution was capable of maintaining stability and viability in concentrated suspensions. 相似文献
12.
The stability and viability of Neisseria gonorrhoeae WP (T4) was tested in a variety of buffers and solutions, many of which are commonly used in gonococcal research. Each solution was tested at room temperature for its ability to maintain stability and viability of gonococci in concentrated suspensions and to maintain viability of gonococci in dilute suspensions. The 14 buffers and solutions tested could be divided into four groups based upon these criteria. Only a few solutions satisfied all three criteria. Of those tested, Gey salt solution and bovine serum albumin (0.01%) and proteose peptone (1%) in saline were the only two in which the gonococci retained viability in dilute suspensions for 25 min. Most of the solutions were not able to maintain viability of gonococci in dilute suspensions, even when the same solution was capable of maintaining stability and viability in concentrated suspensions. 相似文献
13.
The effect of temperature on the interfacial behavior of uracil, thymine and 1,5-dimethyluracil has been used to characterize the thermodynamics of adsorption of these compounds at a mercury electrode-aqueous electrolyte interface. Between 4.5 degrees C and 40 degrees C all three compounds exhibit two adsorption regions. The first, a dilute layer where the molecules adsorb in a flat orientation on the electrode surface. The second, a compact layer where the molecules adsorb in a perpendicular orientation. The thermodynamic constants characterizing formation of these surface layers have been deduced. 相似文献
14.
At low temperature and in a narrow pH-range, the hydroxyl-proton resonance spectra of a range of mono-, di- and oligo-saccharides in dilute aqueous solutions have been resolved. The signals rapidly broaden on raising the temperature and on changing the pH of the solutions. Optimum conditions for obtaining maximum resolution are described and attempts are made to assign the resonances to specific hydroxyl groups. In all cases the resonances for the anomeric hydroxyl-proton occurred at lowest field and the pH value for optimum resolution of these resonances was always lower than that for the other hydroxyl resonances. 相似文献
15.
Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field. 相似文献
16.
A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions. 相似文献
17.
A calorimetric study of the thermal denaturation of bovine serum albumin, RNAase and catalase in concentrated solutions (crystals) has been carried out. The results obtained for RNAase studied within the pH range 2.5-8.5 show that for concentrated solutions there is an interval of pH where, on cooling of the solution which had undergone denaturation, its renaturation is observed. In the case of concentrated and dilute solutions of RNAase these intervals coincide. The study of RNAase under such conditions at various heating rates shows that there is a range of rates in which the process of denaturation of concentrated solutions can be considered as reversible. The dependences of Td and Hd on pH and concentration of solutions have been determined. The denaturation enthalpy of concentrated solutions like in dilute ones, has been found to be independent of the pH of solutions, and the experimentally registered change has been proved to be the result of its dependence on temperature. A new method of determination of protein denaturation enthalpy under the conditions of intensive molecule aggregation is suggested. The forms of irreversibility as appearing in the calorimetric experiment were determined by comparing reversible and irreversible denaturation under continuous and step-heating regimes. It is shown that the decrease in Tmax and the narrowing of the heat absorption peak in the case of decreasing heating rates of protein solutions, observed under certain environmental conditions, results from the irreversibility of the denaturation process. 相似文献
18.
Model-membrane systems composed of liquid-crystalline bicellar phases can be uniaxially oriented with respect to a magnetic field, thereby facilitating structural and dynamics studies of membrane-associated proteins. Here we quantitatively characterize a method that allows the manipulation of the direction of this uniaxial orientation. Bicelles formed from DMPC/DHPC are examined by 31P NMR under variable-angle sample-spinning (VAS) conditions, confirming that the orientation of the liquid-crystalline director can be influenced by sample spinning. The director is perpendicular to the rotation axis when (the angle between the sample-spinning axis and the magnetic field direction) is smaller than the magic angle, and is parallel to the rotation axis when is larger than the magic angle. The new 31P NMR VAS data presented are considerably more sensitive to the orientation of the bicelle than earlier 2H studies and the analysis of the sideband pattern allows the determination of the orientation of the liquid-crystal director and its variation over the sample, i.e., the mosaic spread. Under VAS, the mosaic spread is small if deviates significantly from the magic angle but becomes very large at the magic angle. 相似文献
19.
Harishchandra Ghimire Johnson J. Inbaraj Gary A. Lorigan 《Chemistry and physics of lipids》2009,160(2):98-104
X-band and Q-band electron paramagnetic resonance (EPR) spectroscopic techniques were used to investigate the structure and dynamics of cholesterol containing phospholipid bicelles based upon molecular order parameters (Smol), orientational dependent hyperfine splittings and line shape analysis of the corresponding EPR spectra. The nitroxide spin-label 3-β-doxyl-5-α-cholestane (cholestane) was incorporated into DMPC/DHPC bicelles to report the alignment of bicelles in the static magnetic field. The influence of cholesterol on aligned phospholipid bicelles in terms of ordering, the ease of alignment, phase transition temperature have been studied comparatively at X-band and Q-band. At a magnetic field of 1.25 T (Q-band), bicelles with 20 mol% cholesterol aligned at a much lower temperature (313 K), when compared to 318 K at a 0.35 T field strength for X-band, showed better hyperfine splitting values (18.29 G at X-band vs. 18.55 G at Q-band for perpendicular alignment and 8.25 G at X-band vs. 7.83 G at Q-band for the parallel alignment at 318 K) and have greater molecular order parameters (0.76 at X-band vs. 0.86 at Q-band at 318 K). Increasing cholesterol content increased the bicelle ordering, the bicelle-alignment temperature and the gel to liquid crystalline phase transition temperature. We observed that Q-band is more effective than X-band for studying aligned bicelles, because it yielded a higher ordered bicelle system for EPR spectroscopic studies. 相似文献
20.
We demonstrate by using vibrational circular dichroism (VCD) spectroscopy that it is possible to investigate the chirality of a supramolecular polymeric system in relatively dilute solutions. Chiral C(3)-symmetrical discotic molecules, based on a trialkylbenzene-1,3,5-carboxamide, form supramolecular columnar stacks with a right-handed helical structure in solution due to intermolecular hydrogen bonds. The handedness of the supramolecular chirality is determined using electronic spectroscopy measurements. Under dilute conditions (at 10(-3) M concentrations), it was also possible to probe the hydrogen bonding moieties with IR and VCD spectroscopy on these self-assembled structures. In combination with density functional theory (DFT) calculations, we could verify the preference for a right-handed chirality in the helical stacks and the nonplanar orientation of the carbonyl groups present in the molecule. This chiral arrangement is in agreement with the structure determined for a related benzene-1,3,5-tricarboxamide by X-ray diffraction. Chirality, 2008. (c) 2008 Wiley-Liss, Inc. 相似文献