Lateral pressure in membranes.
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Hydrostatic Pressure↗
Biomedical subjects
D Marsh
Publications and source records attributed to D Marsh.
Peptide models for membrane channels.
Peptides may be synthesized with sequences corresponding to putative transmembrane domains and/or pore-lining regions that are deduced from the primary structures of ion channel proteins. These can then be incorporated into lipid bilayer membranes for structural and functional studies. In addition to the ability to invoke ion channel activity, critical issues are the secondary structures adopted and the mode of assembly of these short transmembrane peptides in the reconstituted systems. The present review concentrates on results obtained with peptides from ligand-gated and voltage-gated ion channels, as well as proton-conducting channels. These are considered within the context of current molecular models and the limited data available on the structure of native ion channels and natural channel-forming peptides.
Alamethicin↗
Lipid chain dynamics and molecular location of diacylglycerol in hydrated binary mixtures with phosphatidylcholine: spin label ESR studies.
The lipid chain motions in hydrated binary mixtures of dimyristoylglycerol (DMG) with dimyristoylphosphatidylcholine (DMPC) have been studied by using ESR spectroscopy of analogues of both components that are spin-labeled at one of eight different positions along the sn-2 chain. The phase diagram of the binary mixtures divides into three separate regions along the composition axis, corresponding to the formation of isothermally melting compounds, with DMPC/DMG stoichiometries of approximately 1:1 and 1:2 mol/mol in the gel phase [Heimburg, T., Wurz, U., & Marsh, D. (1992) Biophys. J. 63, 1369-1378]. In the first region (up to 50 mol % DMG), comparison of the chain flexibility profiles, and the chain profiles of the polarity-dependent isotropic hyperfine coupling constant, of the two different spin-labeled components indicates that DMG is incorporated in the fluid lipid bilayer in a manner similar to that of the host DMPC but is situated approximately two CH2 groups deeper into the hydrophobic interior. At lower contents of DMG, the chain packing is increased by the addition of DMG, whereas at higher DMG contents the lipid chain order decreases rapidly, on reaching the inverted hexagonal phase of the second region of the phase diagram. In the second region of the phase diagram (50-67 mol % DMG), the DMG fits better into the fluid inverted hexagonal phase than into the fluid lamellar phase of the first region and is located only approximately one CH2 group deeper than the corresponding DMPC. In these first two regions of the phase diagram, the ESR spectra of both spin-labeled components display an axial anisotropy that evidences the increasing angular amplitude of motion with position down the chain that is characteristic of liquid crystalline fluid phases. In the third region of the phase diagram (above 67 mol % DMG), the fluid phase consists of isotropically tumbling DMG molecules in which the DMPC molecules are incorporated as inverted micelles as indicated by the residual anisotropic motion of the spin-labeled phosphatidylcholine analogues.
Anisotropy↗
Components of the lateral pressure in lipid bilayers deduced from HII phase dimensions.
The components of the effective internal lateral pressure arising from the lipid headgroups and the lipid chains in bilayer membranes are deduced from the conditions for interfacial equilibrium and the spontaneous bending moments within the component monolayers. The latter are obtained from the intrinsic curvature that is deduced from X-ray diffraction measurements on the corresponding inverted hexagonal (HII) lipid phases.
Lipid Bilayers↗
Mode of insertion of the signal sequence of a bacterial precursor protein into phospholipid bilayers as revealed by cysteine-based site-directed spectroscopy.
The interactions between a bacterial precursor protein and phospholipids in bilayer-based model membrane systems is addressed in this study. The precursor-lipid interactions were assessed from the side of the lipid phase by fluorescence and electron spin resonance spectroscopy, using the precursor of the Escherichia coli outer membrane protein PhoE. The role of the signal sequence, as part of the precursor, in this interaction was investigated by using cysteine-based site-directed spectroscopy. For this purpose, purified cysteine-containing mutants of prePhoE, which were made by site-directed mutagenesis of the signal sequence part and of the mature part, and defined lipids were used. The location of the fluorescently labeled cysteine residues was established by resonance energy transfer and quenching experiments and those of the corresponding spin-labeled cysteine residues by paramagnetic relaxation enhancement. It was demonstrated that precursor-phospholipid interactions exist in model membrane systems and also that these interactions were dependent on the presence of anionic phospholipids and resulted in a deep insertion of (parts of) the precursor into the lipid bilayer. Furthermore, the results with the cysteine mutations in the signal sequence of the precursor indicate that both termini of the signal sequence are located near or at the membrane surface, with only the fluorescence of the labeled cysteines in the signal sequence part being protected against aqueous quenchers. The results demonstrate that, when part of the intact precursor, the signal sequence experiences similar lipid-protein interactions as do isolated signal peptides. They also indicate that the signal sequence inserts entirely as a looped structure into the membrane. In addition, the data also indicate that the mature part of the precursor has an affinity for the membrane.
Amino Acid Sequence↗
Dependence of Saturation-Transfer EPR Intensities on Spin-Lattice Relaxation
The intensities of saturation-transfer EPR (ST-EPR) spectra from nitroxyl spin labels have proved a sensitive means for studying slow exchange processes (both Heisenberg spin exchange and physical/chemical exchange) and weak interactions with paramagnetic ions, via the dependence on the effective spin-lattice relaxation rate (D. Marsh, Appl. Magn. Reson. 3, 53, 1992). The dependences of the second-harmonic EPR absorption intensities detected in phase quadrature with the field modulation (V'2) on the microwave H1 field, and on the effective relaxation times, were studied both theoretically and experimentally. Power-saturation curves and normalized integrated intensities (IST) of the V'2 spectra were determined as a function of the concentration of a spin-labeled phospholipid in lipid membranes and of the concentration of paramagnetic Ni2+ ions in the aqueous phase as a means of varying the effective relaxation times. The results were correlated with progressive-saturation measurements of the double-integrated intensities of the conventional EPR spectra. Intensities of the V'2 spectra were calculated from the Bloch equations incorporating the modulation and microwave fields (K. Halbach, Helv. Phys. Acta 27, 259, 1954), and the results were fitted to the experimental data. The ST-EPR intensities depend approximately linearly on the effective T1, but with a nonzero intercept. On the basis of the theoretical calculations and experimental correlations, relations between IST and T1 are suggested that may improve precision in the application of this alternative form of ST-EPR spectroscopy to biological systems.
Journal Article↗
Epidemiology of adults hospitalized with burns in Karachi, Pakistan.
Burns are a leading cause of adult death in Karachi slums, therefore we reviewed 1 year's logged experience (November 1992 to October 1993) at Karachi's two adult burn units for patient age, sex, burn severity and outcome. Also 47 inpatients were interviewed regarding their circumstances of injury. We grouped these using Haddon's Matrix. The log identified 832 patients. Females (57 per cent) outnumbered males and were younger on average (25.1 vs 27.6 years, P = 0.002). Females had more severe burns than males (57 per cent vs 50 per cent total body surface area (TBSA) burn, P = 0.002). At the unit with outcome data (n = 556), the case fatality was 56 per cent. The estimated adult mortality due to burns in Karachi was 10.2/100 000, 6.8/100 000 and 14.1/100 000 for men and women, respectively. Burns of interviewed patients were most often associated with flames (33/47), but stove bursts caused the most severe injury (52 per cent TBSA). These patients were predominantly young uneducated female houseworkers, clothed in loose attire who were injured during daylight at home around a floor-level stove, unaware of fire safety, and who received no first aid. It was concluded that the high burn severity and case fatality rates demand: (1) preventive measures, such as kitchen sand buckets, safer stove design and placement and education on fire safety and first aid, and (2) risk factor analysis to refine interventions.
Accidents, Home↗
Fourier-transform infrared spectroscopic studies on avidin secondary structure and complexation with biotin and biotin-lipid assemblies.
Fourier-transform infrared studies have been carried out to investigate the secondary structure and thermal stability of hen egg white avidin and its complexes with biotin and with a biotinylated lipid derivative, N-biotinyl dimyristoyl phosphatidylethanolamine (DMBPE) in aqueous dispersion. Analysis of the amide I stretching band of avidin yielded a secondary structural content composed of approximately 66% beta-sheet and extended structures, with the remainder being attributed to disordered structure and beta-turns. Binding of biotin or specific association with the biotinylated lipid DMBPE did not result in any appreciable changes in the secondary structure content of the protein, but a change in hydrogen bond stability of the beta-sheet or extended chain regions was indicated. The latter effect was enhanced by surface interactions in the case of the biotin-lipid assemblies, as was demonstrated by electrostatic binding to a nonspecific negatively charged lipid. Difference spectra of the bound biotin implicated a direct involvement of the ureido moiety in the ligand interaction that was consistent with hydrogen bonding to amino acid residues in the avidin protein. It was found that complexation with avidin leads to a decrease in bond length of the biotin ureido carbonyl group that is consistent with a reduction of sp3 character of the C-O bond when it is hydrogen bonded to the protein. Studies of the temperature dependence of the spectra revealed that for avidin alone the secondary structure was unaltered up to approximately 75 degrees C, above which the protein undergoes a highly cooperative transition to an unfolded state with concomitant loss of ordered secondary structure. The complexes of avidin with both biotin and membrane-bound DMBPE lipid assemblies display a large increase in thermal stability compared with the native protein.
Avidin↗
Fluorescence-quenching study of percolation and compartmentalization in two-phase lipid bilayers.
Fluorescence quenching of a lipid-labeled fluorophore by a lipid spin-labeled quencher has been studied experimentally in two-component, two-phase phosphatidylcholine bilayers to examine the effect of phase connection and disconnection on quenching. Both fluorophore and quencher prefer the fluid phase. At the percolation threshold, the point at which the fluid phase becomes subdivided into may small disconnected domains, the quenching drops abruptly. This decrease in quenching is a function of the fluid-phase fraction and is due to the heterogeneous distribution of fluorophores and quenchers over the fluid-phase domains. Computer simulations of the system were carried out with a triangular lattice divided into closed compartments of variable size and reactant occupancy. The simulations demonstrate that the degree of quenching is reduced in the disconnected systems and that the reduction is correlated with the size of the disconnected domains. The combination of experimental data with simulations leads to the conclusion that at constant temperature the size of fluid-phase domains, nfluid, in the region of the coexistence of the fluid and gel phases is proportional to the fluid fraction, Xfluid. This is in a qualitative agreement with a previous electron spin resonance study of interlipid spin-spin interactions in the same two-component, two-phase bilayer system.
1,2-Dipalmitoylphosphatidylcholine↗
Dynamic chain conformations in dimyristoyl glycerol-dimyristoyl phosphatidylcholine mixtures. 2H-NMR studies.
The dynamic molecular lipid chain conformations in fully hydrated dimyristoyl phosphatidylcholine (DMPC)-dimyristoyl glycerol (DMG) mixtures have been investigated by 2H-NMR spectroscopy of the individual lipid components, the sn-2 chains of which were perdeuterated or, in the case of DMG, specifically deuterated at the C-2 position. Mixtures of compositions corresponding to the three different regions of the binary phase diagram in which the fluid phase is lamellar (DMPC:DMG 70:30 mol/mol), inverted hexagonal (DMPC:DMG 45:55 and 40:60 mol/mol), or isotropic (DMPC:DMG 20:80 mol/mol) were investigated. The gel phase in all three regions of the phase diagram has a lamellar structure, with the lipid chains rotating about the molecular long axis but executing only limited angular excursions. In the fluid lamellar phase of the 70:30 mol/mol DMPC-DMG mixture the profile of segmental chain flexibility is similar to that in single-component phospholipid bilayers and is characterized by an order parameter plateau for both lipid components. The chain order of the DMPC component is greater than in bilayers of DMPC alone and is also greater than that of the DMG component. In the inverted hexagonal phase of the 45:55 mol/mol DMPC-DMG mixture the chain flexibility profile is characterized by more widely spaced segmental order parameters off the plateau region. The intrinsic degree of chain order in the inverted hexagonal phase is less than in the lamellar phase of the 70:30 mol/mol mixture, and the difference in chain order between the DMPC and DMG components is reduced relative to that in the lamellar phase. The unique conformational features at the C-2 position of the sn-2 chain that characterize bilayers of diacyl phospholipids are found also for the diacylglycerol molecules in the fluid lamellar phase and most probably also in the inverted hexagonal phase. The DMG molecules are therefore integrated in the membrane (or nonlamellar lipid phase) in a configuration that is similar to that of the phospholipids and different from the crystal structure of diacylglycerols.
Deuterium↗
Intrinsic curvature in normal and inverted lipid structures and in membranes.
The intrinsic or spontaneous radius of curvature, R(o), of lipid monolayer assemblies is expressed in terms of a lipid molecular packing parameter, V/AI, for various geometries. It is shown that the equivalent lipid length, 1, in inverted hexagonal (HII) phases, defined by a cylindrical shell of equal total lipid volume, yields an expression for R o identical to that for inverted cylindrical micelles (or, equivalently, HII phases in the presence of excess hydrocarbon). This identity is used to obtain values of the effective packing parameter for various phosphatidylethanolamines. The temperature dependence of the intrinsic radius of curvature is predicted to be negative and to be considerably greater than that for the lipid length in nearly all cases. The thermal expansion coefficient is not constant but is found to vary, depending on the value of the lipid packing parameter. A possible addition rule is constructed for the intrinsic radius of curvature of lipid mixtures, based on the linear additivity of the effective molecular volumes, V, and molecular areas, A. This relation is found to hold for mixtures of dioleoyl phosphatidylcholine (DOPC) with dioleoyl phosphatidylethanolamine, and a value of R(o) of > or = 9 A (V/AI = 1.08) is obtained for DOPC. The energetics of the intrinsic curvature and lamellar-nonlamellar transitions are also discussed within the framework of the model.
Kinetics↗
Cloning and expression of complementary DNA coding for an allergen with common antibody-binding specificities with three allergens of the house dust mite Blomia tropicalis.
The mite Blomia tropicalis is a potent source of allergens in tropical and subtropical regions. So far, most of these allergens have only been studied by immunoblotting. To characterize them at the molecular level, a lambda gt11 complementary DNA library was constructed from messenger RNA isolated from whole B. tropicalis mites. This library was screened by using pooled sera from patients allergic to B. tropicalis in a plaque IgE radioimmunoassay. A B. tropicalis IgE-positive clone (Bt-M) was selected for immunologic studies. After subcloning into pBluescript (Stratagene, La Jolla, Calif.), it produced a sequence of 310 bp, with a probable amino acid sequence of 72 residues for the expressed peptide. The recombinant protein was transferred to nitrocellulose filters and probed with 100 sera from patients allergic to B. tropicalis. Forty-seven percent of sera reacted with the recombinant allergen. Immunoblottings performed with allergic serum and B. tropicalis-affinity-purified IgE demonstrated that the recombinant protein shares allergenic epitopes with the 11-13, 14, and 16 kd native allergens of B. tropicalis, which are known to be important allergens of this mite.
Adolescent↗
Front-line management of pulmonary tuberculosis: an analysis of tuberculosis and treatment practices in urban Sindh, Pakistan.
SETTING: Karachi and Hyderabad, Pakistan. OBJECTIVE: To describe the level and quality of tuberculosis (TB) case management by non-TB control program (TCP) physicians in urban Sindh, Pakistan. DESIGN: We interviewed 152 adults with pulmonary TB confirmed by Karachi's TB control program regarding the initial management of their TB symptoms before entering the TCP. We also surveyed 65 general practitioners (GPs) attending continuing education seminars with a multiple choice test to assess their management of suspected pulmonary TB. We compared both results to guidelines from the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD). RESULTS: Eighty percent (122/152) of patients first sought GPs. Only 14% of GPs performed any sputum test. At most, 17 (40%) of the 42 patients recalling their GP's treatment, received the recommended 4-drug regimen. However, 68% (45/65) of surveyed GPs chose correct treatment from a multiple choice format. But their initial laboratory investigations, follow-up, and treatment cessation criteria (9%, 9-31%, and 11% correct, respectively) demonstrated under-utilization of sputum tests and over-reliance on unhelpful tests. CONCLUSIONS: GPs first saw most of these TCP patients, but their weak management likely hinders TB control. A partnership between TB control programs and GPs could improve case management and hasten TB control.
Adolescent↗
Dose reduction in double contrast barium enema by use of low fluoroscopic current.
Under standard conditions double contrast barium enema (DCBE) was carried out on a selected patient cohort using either standard (1.5 mA) or low (0.5 mA) fluoroscopic current. A statistically significant (p < or = 0.01) reduction in total dose was achieved by use of low screening current. This reduction was of the order of 40%. Fluoroscopic image quality was analysed both objectively and subjectively, and was found not to be significantly degraded when the lower current was used. On blinded evaluation all examinations were found to be of diagnostic quality. The use of low fluoroscopic current results in a reduction in patient dose, without compromising fluoroscopic image quality or diagnostic standard of the examination. Low fluoroscopic current technique has been adopted as the standard in our department.
Barium Sulfate↗
Membrane assembly studied by spin-label electron spin resonance.
Conventional electron spin resonance (ESR) of spin-labelled lipids and saturation transfer ESR of spin-labelled proteins are used to study lipid-protein interactions and the mobility of integral proteins, respectively, both in biological membranes and in reconstituted lipid-protein systems. Conventional ESR spectra reveal two spin-labelled lipid populations, the mobility of one of which is hindered by direct interaction with the integral membrane proteins. The proportion of the latter component increases with increasing protein content and with increasing selectivity of the lipid species for the protein. The two-component spectra are quantitated by spectral subtraction and addition, and by simulation using the exchange-coupled Bloch equations. Lipid exchange rates at the protein interface obtained by simulation are found to be consistent with fast exchange found by 2H NMR on similar systems and to reflect the lipid selectivity observed by ESR. Protein-reactive covalent spin labels have been used to study the rotational diffusion and aggregation states of membrane proteins via saturation transfer ESR. The integral protein rotation is uniaxial to the first approximation and the anisotropic motion is analyzed to obtain the principal component of the diffusion tensor. The latter is sensitively dependent on the cross-sectional dimensions of the protein in the membrane, and hence on its state of assembly. A variety of new experiments based on the power saturation properties of the spin-labelled components are also used to determine lipid exchange rates, protein translational diffusion rates, and the location and penetration of proteins in membranes.
Diffusion↗
Interaction of alpha-lactalbumin with phosphatidylglycerol. Influence of protein binding on the lipid phase transition and lipid acyl chain mobility.
The mobility of spin-labeled lipids has been studied in dioleoyl and dimyristoyl phosphatidylglycerol bilayers and in their complexes with alpha-lactalbumin at pH 4.0, by using electron spin resonance (ESR) spectroscopy. The ESR spectra of phosphatidylglycerol spin-labeled at position 5 of the sn-2 chain indicate that association of alpha-lactalbumin with dimyristoyl phosphatidylglycerol bilayers increases the chain mobility at temperatures in the lipid gel phase, restricts the chain mobility at temperatures corresponding to the lipid fluid phase, and abolishes the cooperative lipid chain-melting transition. The ESR spectra of phosphatidylglycerols spin-labeled at eight different positions in the sn-2 chain show that binding of alpha-lactalbumin to dioleoyl phosphatidylglycerol bilayers at pH 4.0 causes a motional restriction throughout the full length of the lipid acyl chain. For phosphatidylglycerols spin-labeled at the terminal methyl end of the chains, a population of motionally restricted lipids that directly contacts membrane penetrant portions of the protein is detected. This population corresponds to 6.3 +/- 0.7 lipids/alpha-lactalbumin at saturation binding, and the high degree of motional restriction (maximum hyperfine splitting approximately 60 G) suggests that the protein may traverse the lipid bilayer. A small selectivity of phosphatidylglycerol over zwitterionic phospholipids for interaction with alpha-lactalbumin is found at subsaturating levels of binding at pH 4.0. Binding of alpha-lactalbumin also strongly restricts the motion of lipids spin-labeled in the polar head group region. These results are of direct relevance to the insertion and translocation of a protein in the molten globule state across lipid membranes.
Electron Spin Resonance Spectroscopy↗
Insertion of diphtheria toxin in lipid bilayers studied by spin label ESR.
The pH dependence of the insertion of diphtheria toxin into bilayers of dioleoylphosphatidylglycerol (DOPG) has been studied by using ESR spectroscopy of spin-labeled phosphatidylglycerol with the reporter group at either the 5-position or the 14-position of the sn-2 chain (5-PGSL and 14-PGSL, respectively). At neutral pH, addition of diphtheria toxin has little effect on the ESR spectra of either spin label in large unilamellar vesicles of DOPG. At acidic pH, the outer hyperfine splitting of the 5-PGSL label is increased, and a second component corresponding to lipids whose chain motion is selectively restricted appears in the spectra of the 14-PGSL label, in the presence of diphtheria toxin. The motionally restricted component of 14-PGSL has a large outer hyperfine splitting (2Amax approximately 61 G) and corresponds to spin-labeled lipids the chains of which are in direct contact with the membrane-penetrant part of the inserted toxin. This restricted component is present, although to a lesser extent, in vesicles containing 90% of the zwitterionic lipid dioleoylphosphatidylcholine and displays a limited selectivity for negatively charged relative to zwitterionic spin-labeled phospholipids. The fraction of lipids which are motionally restricted by the toxin increases with decreasing pH, titrating in DOPG vesicles with an apparent pKa of approximately 6.1. The outer hyperfine splitting of the 5-PGSL label titrates with an apparent pKa of approximately 5.5, suggesting that this might be preferentially sensitive to a later stage in the insertion of the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
Diphtheria Toxin↗
Lipid-protein interactions and assembly of the 16-kDa channel polypeptide from Nephrops norvegicus. Studies with spin-label electron spin resonance spectroscopy and electron microscopy.
The assembly of 16-kDa polypeptide channel units in membranes from the hepatopancreas of Nephrops norvegicus has been studied both by electron microscopy and by the lipid--protein interactions reported with spin-labeled lipids. Membranes prepared by extraction with N-lauroylsarcosine and Triton X-100 have a low lipid/protein ratio (ca. 4-6.5 phospholipids and 1 cholesterol per 16-kDa monomer), and those prepared by alkaline extraction have a higher lipid/protein ratio (ca. 12-16 phospholipids and 3.5-4 cholesterols per 16-kDa monomer). In the membranes extracted with detergents, the protein is assembled in membrane sheets as hexagonally packed hexameric complexes, whereas the alkali-extracted preparations consist of closed vesicles in which the channel complexes are near randomly distributed. The electron spin resonance (ESR) spectra from lipids spin-labeled at the C-14 position of the (sn-2) chain show lower mobility for the membranes extracted with N-lauroylsarcosine than for the alkaline-extracted membranes. At higher temperatures, the ESR spectra reveal a population of lipids whose mobility is restricted by direct interaction with the intramembranous sections of the channel assemblies. The population of protein-associated spin-labeled phosphatidylcholine in the alkali-extracted membranes corresponds to 4-5 phospholipid molecules plus 1 cholesterol molecule per 16-kDa polypeptide monomer.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗