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1.
Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date. 相似文献
2.
Julie E. Norville Deborah F. Kelly Thomas F. Knight Jr. Angela M. Belcher Thomas Walz 《Journal of structural biology》2007,160(3):313
Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date. 相似文献
3.
Polymer-cushioned bilayers. I. A structural study of various preparation methods using neutron reflectometry.
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J Y Wong J Majewski M Seitz C K Park J N Israelachvili G S Smith 《Biophysical journal》1999,77(3):1445-1457
This neutron reflectometry study evaluates the structures resulting from different methods of preparing polymer-cushioned lipid bilayers. Four different techniques to deposit a dimyristoylphosphatidylcholine (DMPC) bilayer onto a polyethylenimine (PEI)-coated quartz substrate were examined: 1) vesicle adsorption onto a previously dried polymer layer; 2) vesicle adsorption onto a bare substrate, followed by polymer adsorption; and 3, 4) Langmuir-Blodgett vertical deposition of a lipid monolayer spread over a polymer-containing subphase to form a polymer-supported lipid monolayer, followed by formation of the outer lipid monolayer by either 3) horizontal deposition of the lipid monolayer or 4) vesicle adsorption. We show that the initial conditions of the polymer layer are a critical factor for the successful formation of our desired structure, i.e., a continuous bilayer atop a hydrated PEI layer. Our desired structure was found for all methods investigated except the horizontal deposition. The interaction forces between these polymer-supported bilayers are investigated in a separate paper (Wong, J. Y., C. K. Park, M. Seitz, and J. Israelachvili. 1999. Biophys. J. 77:1458-1468), which indicate that the presence of the polymer cushion significantly alters the interaction potential. These polymer-supported bilayers could serve as model systems for the study of transmembrane proteins under conditions more closely mimicking real cellular membrane environments. 相似文献
4.
The lung surfactant proteins SP-B and SP-C are pivotal for fast and reversible lipid insertion at the air/liquid interface, a prerequisite for functional lung activity. We used a model system consisting of a preformed monolayer at the air/liquid interface supplemented with surfactant protein SP-B or SP-C and unilamellar vesicles injected into the subphase of a film balance. The content of SP-B or SP-C was similar to that found in lung lavage. In order to elucidate distinct steps of lipid insertion, we measured the time-dependent pressure increase as a function of the initial surface pressure, the temperature and the phosphatidylglycerol content by means of surface tension measurements and scanning force microscopy (SFM). The results of the film balance study are indicative of a two-step mechanism in which initial adsorption of vesicles to the protein-containing monolayer is followed by rupture and integration of lipid material. Furthermore, we found that vesicle adsorption on a preformed monolayer supplemented with SP-B or SP-C is strongly enhanced by negatively charged lipids as provided by DPPG and the presence of Ca2+ ions in the subphase. Hence, long-range electrostatic interactions are thought to play an important role in attracting vesicles to the surface, being the initial step in replenishment of lipid material. While insertion into the monolayer is independent of the type of protein SP-B or SP-C, initial adsorption is faster in the presence of SP-B than SP-C. We propose that the preferential interaction between SP-B and negatively charged DPPG leads to accumulation of negative charges in particular regions, causing strong adhesion between DPPG-containing vesicles and the monolayer mediated by Ca2+ ions, which eventually causes flattening and rupture of attached liposomes as observed by in situ SFM. 相似文献
5.
Recognition processes at a functionalized lipid surface observed with molecular resolution. 总被引:1,自引:0,他引:1
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The specific binding of proteins to functionalized lipid monolayers on aqueous subphases was characterized by neutron reflectivity and fluorescence microscopy measurements. Due to the high affinity and high specificity of their noncovalent interaction, streptavidin (SA) and biotin (vitamin H) were chosen as a model system to investigate the structural characteristics of a recognition process on a molecular length scale. Changes in the neutron reflection from the surfaces of NaCl aqueous (H2O or D2O) protein solutions (10(-8) M SA) were used to monitor the interaction of the protein with a monolayer of a biotinylated lipid in situ. Refinement of the reflectivity data and independent fluorescence microscopic observation of the interface using FITC-labeled SA showed that the protein forms macroscopically homogeneous (and presumably crystalline) domains covering a large portion of the surface. Moreover, the neutron reflection experiments clearly showed the formation of a monomolecular protein layer with an effective thickness, dp = 43.7 +/- 2 A. The area per protein molecule occupied in the film was A0 = 2860 +/- 200 A2 and nw = 260 +/- 100 water molecules were associated with each protein molecule. Quantitative binding was found to occur at biotin surface concentrations as low as 1 molecule/1,250 A2 (compared with approximately 1 molecule/40 A2 for dense packing). This study demonstrates the application of a promising new tool for the systematic investigation of molecular recognition processes in protein/lipid model systems. 相似文献
6.
Sensitive detection of protein adsorption to supported lipid bilayers by frequency-dependent capacitance measurements and microelectrophoresis 总被引:2,自引:0,他引:2
In the first part, we report experiments which enable the sensitive detection of protein adsorption to lipid bilayers deposited onto chromium electrodes on glass substrates by frequency-dependent capacitance measurements. The sensitivity of the present type of sensor (better than 0.3 nm average protein layer thickness) is at least equivalent to that of ellipsometry. A high specific resistance of the supported bilayer of (1-5).10(5) omega.cm2 is achieved by deposition of a tightly packed (crystalline) cadmium arachidate monolayer in contact with the substrate, whereas the outer monolayer can be more loosely packed (fluid phase or state of fluid-solid coexistence) which is essential for the incorporation of receptors. In the present work, charged lipids are incorporated as nonspecific receptors for polylysine and cytochrome c. The capacitance measurements provide a very sensitive test of the tightness and the long-time stability of the supported bilayers and, in combination with ellipsometric thickness measurements, enable estimations of dielectric properties of protein layers (such as the permittivity). In the second part, we report first electrophoresis experiments in asymmetric bilayers on substrates which enable simultaneous measurements of lateral diffusion coefficients and frictional coefficients between monolayers. The potential application of the electrophoretic effect for the differentiation between different receptors and the amplification of signals in biosensors is discussed. 相似文献
7.
Teboul D Beaufils S Taveau JC Iatmanen-Harbi S Renault A Venien-Bryan C Vie V Lacapere JJ 《Biochimica et biophysica acta》2012,1818(11):2791-2800
Translocator protein TSPO is a membrane protein highly conserved in evolution which does not belong to any structural known family. TSPO is involved in physiological functions among which transport of molecules such as cholesterol to form steroids and bile salts in mammalian cells. Membrane protein structure determination remains a difficult task and needs concomitant approaches (for instance X-ray- or Electron-crystallography and NMR). Electron microscopy and two-dimensional crystallization under functionalized monolayers have been successfully developed for recombinant tagged proteins. The difficulty comes from the detergent carried by membrane proteins that disrupt the lipid monolayer. We identified the best conditions for injecting the histidine tagged recombinant TSPO in detergent in the subphase and to keep the protein stable. Reconstituted recombinant protein into a lipid bilayer favors its adsorption to functionalized monolayers and limits the disruption of the monolayer by reducing the amount of detergent. Finally, we obtained the first transmission electron microscopy images of recombinant mouse TSPO negatively stained bound to the lipid monolayer after injection into the subphase of pre-reconstituted TSPO in lipids. Image analysis reveals that circular objects could correspond to an association of at least four monomers of mouse TSPO. The different amino acid compositions and the location of the polyhistidine tag between bacterial and mouse TSPO could account for the formation of dimer versus tetramer, respectively. The difference in the loop between the first and second putative transmembrane domain may contribute to distinct monomer interaction, this is supported by differences in ligand binding parameters and biological functions of both proteins. 相似文献
8.
Atomic force microscopy has been used to follow in real time the adsorption from solution of two of the gliadin group of wheat seed storage proteins onto hydrophilic (mica) and hydrophobic (graphite) surfaces. The liquid cell of the microscope was used initially to acquire images of the substrate under a small quantity of pure solvent (1% acetic acid). Continuous imaging as an injection of gliadin solution entered the liquid cell enabled the adsorption process to be followed in situ from zero time. For ω‐gliadin, a monolayer was formed on the mica substrate during a period of ~2000 s, with the protein molecules oriented in parallel to the mica surface. In contrast, the ω‐gliadin had a relatively low affinity for the graphite substrate, as demonstrated by slow and weak adsorption to the surface. With γ‐gliadin, random deposition onto the mica surface was observed forming monodispersed structures, whereas on the graphite surface, monolayer islands of protein were formed with the protein molecules in a perpendicular orientation. Sequential adsorption experiments indicated strong interactions between the two proteins that, under certain circumstances, caused alterations to the surface morphologies of preadsorbed species. The results are relevant to our understanding of the interactions of proteins within the hydrated protein bodies of wheat grain and how these determine the processing properties of wheat gluten and dough. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 74–84, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
9.
Puroindolines, cationic and cystine-rich low molecular weight lipid binding proteins from wheat seeds, display unique foaming properties and antimicrobial activity. To unravel the mechanism involved in these properties, the interaction of puroindoline-a (PIN-a) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers was studied by coupling Langmuir-Blodgett and imaging techniques. Compression isotherms of PIN-a/phospholipid monolayers and adsorption of PIN-a to lipid monolayers showed that the protein interacted strongly with phospholipids, especially with the anionic DPPG. The electrostatic contribution led to the formation of a highly stable lipoprotein monolayer. Confocal laser scanning microscopy and atomic force microscopy showed that PIN-a was mainly inserted in the liquid-expanded phase of the DPPC, where it formed an aggregated protein network and induced the fusion of liquid-condensed domains. For DPPG, the protein partitioned in both the liquid-expanded and liquid-condensed phases, where it was aggregated. The extent of protein aggregation was related both to the physical state of phospholipids, i.e., condensed or expanded, and to the electrostatic interactions between lipids and PIN-a. Aggregation of PIN-a at air-liquid and lipid interfaces could account for the biological and technological properties of this wheat lipid binding protein. 相似文献
10.
Streptavidin binding to biotinylated lipid layers on solid supports. A neutron reflection and surface plasmon optical study.
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Neutron reflection and surface plasmon optical experiments have been performed to evaluate structural data of the interfacial binding reaction between the protein streptavidin and a solid-supported lipid monolayer partly functionalized by biotin moieties. Since both experimental techniques operate in a total internal reflection geometry at a substrate/solution interface, identical sample architectures allow for a direct comparison between the results obtained with these two recently developed methods. It is found that a monomolecular layer of dipalmitoyllecithin doped with 5 mol% of a biotinylated-phosphatidylethanolamine shows a thickness of d1 approximately (3.4 +/- 0.5) nm. Binding of streptavidin to the biotin groups results in an overall layer thickness of d = (5.9 + 0.5) nm that demonstrates the formation of a well-ordered protein monolayer with the (biotin+spacer) units of the functionalized lipids being fully embedded into the binding pocket of the proteins. It is demonstrated by model calculations that a more detailed picture of the internal structure of this supramolecular assembly can only be obtained if one uses deuterated lipid molecules, thus generating a high contrast between individual layers. 相似文献
11.
Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A
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Zuo YY Tadayyon SM Keating E Zhao L Veldhuizen RA Petersen NO Amrein MW Possmayer F 《Biophysical journal》2008,95(6):2779-2791
Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism. 相似文献
12.
X-ray scattering with momentum transfer in the plane of membrane. Application to gramicidin organization. 总被引:1,自引:1,他引:0
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We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments. 相似文献
13.
Nicolas Coudray Gilles Hermann Daniel Caujolle-Bert Argyro Karathanou Françoise Erne-Brand Jean-Luc Buessler Pamela Daum Juergen M. Plitzko Mohamed Chami Urs Mueller Hubert Kihl Jean-Philippe Urban Andreas Engel Hervé-W. Rémigy 《Journal of structural biology》2011,173(2):365-374
We have built and extensively tested a tool-chain to prepare and screen two-dimensional crystals of membrane proteins by transmission electron microscopy (TEM) at room temperature. This automated process is an extension of a new procedure described recently that allows membrane protein 2D crystallization in parallel (Iacovache et al., 2010). The system includes a gantry robot that transfers and prepares the crystalline solutions on grids suitable for TEM analysis and an entirely automated microscope that can analyze 96 grids at once without human interference. The operation of the system at the user level is solely controlled within the MATLAB environment: the commands to perform sample handling (loading/unloading in the microscope), microscope steering (magnification, focus, image acquisition, etc.) as well as automatic crystal detection have been implemented. Different types of thin samples can efficiently be screened provided that the particular detection algorithm is adapted to the specific task. Hence, operating time can be shared between multiple users. This is a major step towards the integration of transmission electron microscopy into a high throughput work-flow. 相似文献
14.
《BBA》1985,810(1):73-83
Studies on monomolecular layers of phospholipids containing the antenna protein B800–850 (LHCP) and in some cases additionally the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides are reported. Information on monolayer preparation as well as on protein/lipid and protein/protein interaction is obtained by means of fluorescence spectroscopy and microscopy at the air/water interface in combination with film balance experiments. It is shown that a homogeneous distribution of functional proteins can be achieved. This can be transformed into a regular pattern-like distribution by inducing a phospholipid phase transition. Although the LHCP preferentially partitions into the fluid lipid phase, it decreases the lateral pressure necessary to crystallize the lipid. This is probably due to an increase in order of the fluid phase. A pressure-induced conformation change of the LHCP is detected via a drastic change in fluorescence yield. A highly efficient energy transfer from LHCP to the reaction center is observed. This proves the quantitative reconstitution of both types of proteins and indicates protein aggregation also in the monolayer. 相似文献
15.
Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO2 substrate. The layer thickness of 32 Å was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 Å. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity. 相似文献
16.
Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability
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Ding J Takamoto DY von Nahmen A Lipp MM Lee KY Waring AJ Zasadzinski JA 《Biophysical journal》2001,80(5):2262-2272
Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome. 相似文献
17.
Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes. 总被引:3,自引:3,他引:0
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The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally, it is found that the binding capacity for the native protein is increased at low ionic strength to a value that is greater than that for complete surface coverage, and that corresponds more closely to neutralization of the effective charge (determined from the ionic strength dependence), rather than of the total net charge, on the protein. Electron spin resonance experiments with spin-labeled lipids indicate that this different mode of binding arises from a penetration or disturbance of the bilayer surface by the protein that may alleviate the effects of in-plane interactions under conditions of strong binding. 相似文献
18.
We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level. 相似文献
19.
In order to clarify the structural relationship between lipid monolayer and bilayer membranes, physical states of these membranes are discussed from their energetic points of view. It is concluded that the monolayer formed at the oil/water interface is a proper model system to represent the physical state of half of a bilayer in its liquid crystalline state. The theoretical prediction is confirmed by the monolayer surface tension measurements and the bilayer conductance experiments with water soluble (extrinsic) proteins. It is also deduced that the surface pressure of the bilayer in the liquid crystalline state is quite high, about 45 dyn/cm, and the interaction of cytochrome c with the bilayer is mainly electrostatic at the bilayer membrane periphery. 相似文献
20.
The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning. 相似文献