Biomedical subjects

D Marsh

Publications and source records attributed to D Marsh.

At least 37 records · Page 2Linked to original sources

PubMed2001

Lipid membrane expansion and micelle formation by polymer-grafted lipids: scaling with polymer length studied by spin-label electron spin resonance.

Spin-label electron spin resonance (ESR) spectroscopy and auxiliary optical density measurements are used to study lipid dispersions of N-poly(ethylene glycol)-dipalmitoyl phosphatidylethanolamine (PEG:5000-DPPE) mixed with dipalmitoyl phosphatidylcholine (DPPC). PEG:5000-DPPE bears a large hydrophilic polymer headgroup (with approximately 114 oxyethylene monomers) and is commonly used for steric stabilization of liposomes used in drug delivery. Comparison is made with results from mixtures of DPPC with polymer lipids bearing shorter headgroups (approximately 45 and 8 oxyethylene monomers). ESR spectra of phosphatidylcholine spin-labeled on the 5-C atom position of the sn-2 chain are shown to reflect the area expansion of the lipid membranes by the lateral pressure exerted in the polymer brush, in a way that is consistent with theory. The lipid chain packing density at the onset of micelle formation is the same for all three PEG-lipids, although the mole fraction at which this occurs differs greatly. The mole fraction at onset scales inversely with the size of the polymer headgroup, where the experimental exponent of 0.7 is close to theoretical predictions (viz. 0.55-0.6). The mole fraction of PEG-lipid at completion of micelle formation is more weakly dependent on polymer size, which conforms with theoretical predictions. At high mole fractions of PEG:5000-DPPE the dependence of lipid packing density on mole fraction is multiphasic, which differs qualitatively from the monotonic decrease in packing density found with the shorter polymer lipids. Lipid spin-label ESR is an experimental tool that complements theoretical analysis using polymer models combined with the lipid equation of state.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

Tilt, twist, and coiling in beta-barrel membrane proteins: relation to infrared dichroism.

The x-ray coordinates of beta-barrel transmembrane proteins from the porins superfamily and relatives are used to calculate the mean tilt of the beta-strands and their mean local twist and coiling angles. The 13 proteins examined correspond to beta-barrels with 8 to 22 strands, and shear numbers ranging from 8 to 24. The results are compared with predictions from the model of Murzin, Lesk, and Chothia for symmetrical regular barrels. Good agreement is found for the mean strand tilt, but the twist angles are smaller than those for open beta-sheets and beta-barrels with shorter strands. The model is reparameterised to account for the reduced twist characteristic of long-stranded transmembrane beta-barrels. This produces predictions of both twist and coiling angles that are in agreement with the mean values obtained from the x-ray structures. With the optimized parameters, the model can then be used to determine twist and coiling angles of transmembrane beta-barrels from measurements of the amide band infrared dichroism in oriented membranes. Satisfactory agreement is obtained for OmpF. The strand tilt obtained from the x-ray coordinates, or from the reparameterised model, can be combined with infrared dichroism measurements to obtain information on the orientation of the beta-barrel assembly in the membrane.

Bacterial Outer Membrane Proteins↗

PubMed2001

Application of electron spin resonance for investigating peptide-lipid interactions, and correlation with thermodynamics.

Peptide-lipid interactions can be investigated with spin-labelled lipid probes by using electron spin resonance (ESR) methods that have been developed for studying lipid-protein interactions with both integral and peripheral membrane proteins and also with surface-binding proteins that additionally penetrate the membrane. This approach has the advantage that a direct comparison can be made with the databank of ESR results from the various types of membrane protein. The appropriateness of the peptides as models for membrane proteins, or for their specific segments, can then be assessed. Further, differences in behaviour can be readily identified, as for example in the case of surface-active cytolytic or fusogenic peptides. Comparison with thermodynamic predictions for membrane insertion provides a useful adjunct to the spin-label method.

Amino Acid Sequence↗

PubMed2001

Constrained modeling of spin-labeled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer.

The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment. A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label. Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants. The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model. A single subclass of structure has been identified that best satisfies these criteria simultaneously. The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems. It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments.

Amino Acid Substitution↗

PubMed2000

Effect of angiotensin II non-peptide AT(1) antagonist losartan on phosphatidylethanolamine membranes.

Losartan was found to affect both the thermotropic behavior and molecular mobility of dimyristoyl- and dipalmitoyl-phosphatidylcholine membranes (Theodoropoulou and Marsh, Biochim. Biophys. Acta 1461 (1999) 135-146). At low concentrations, the antagonist is located close to the interfacial region of the phosphatidylcholine bilayer while at high mole fractions it inserts deeper in the bilayers. In the present study, we investigated the interactions of losartan with phosphatidylethanolamine membranes using differential scanning calorimetry (DSC), electron spin resonance (ESR) and 31P nuclear magnetic resonance (NMR) spectroscopy. DSC showed that the antagonist affected the thermotropic transitions of dimyristoyl-, dipalmitoyl- and dielaidoyl-phosphatidylethanolamine membranes (DMPE, DPPE and DEPE, respectively). ESR spectroscopy showed that the interaction of losartan with phosphatidylethanolamine membranes is more superficial than in the case of phosphatidylcholine bilayers. Additionally, losartan increased the spin-spin broadening of 12-PESL spin labels in the gel phase of DMPE and DPPE membranes, while in the case of DEPE membranes the opposite effect was observed. (31)P-NMR showed that the antagonist stabilizes the fluid lamellar phase of DEPE membranes relative to the hexagonal H(II) phase. Our results show that losartan affects the thermotropic behavior of phosphatidylethanolamine membranes, while the molecular mobility of the membranes is not affected greatly. Furthermore, its interactions with phosphatidylethanolamine membranes are more superficial than with phosphatidylcholine bilayers.

Angiotensin Receptor Antagonists↗

PubMed2000

Racial disparities in access to renal transplantation--clinically appropriate or due to underuse or overuse?

BACKGROUND: Despite abundant evidence of racial disparities in the use of surgical procedures, it is uncertain whether these disparities reflect racial differences in clinical appropriateness or overuse or underuse of inappropriate care. METHODS: We performed a literature review and used an expert panel to develop criteria for determining the appropriateness of renal transplantation for patients with end-stage renal disease. Using data from five states and the District of Columbia on patients who had started to undergo dialysis in 1996 or 1997, we selected a random sample of 1518 patients (age range, 18 to 54 years), stratified according to race and sex. We classified the appropriateness of patients as data on candidates for transplantation and analyzed rates of referral to a transplantation center for evaluation, placement on a waiting list, and receipt of a transplant according to race. RESULTS: Black patients were less likely than white patients to be rated as appropriate candidates for transplantation according to appropriateness criteria based on expert opinion (71 blacks [9.0 percent] vs. 152 whites [20.9 percent]) and were more likely to have had incomplete evaluations (368 [46.5 percent] vs. 282 [38.8 percent], P<0.001 for the overall chi-square). Among patients considered to be appropriate candidates for transplantation, blacks were less likely than whites to be referred for evaluation, according to the chart review (90.1 percent vs. 98.0 percent, P=0.008), to be placed on a waiting list (71.0 percent vs. 86.7 percent, P=0.007), or to undergo transplantation (16.9 percent vs. 52.0 percent, P<0.001). Among patients classified as inappropriate candidates, whites were more likely than blacks to be referred for evaluation (57.8 percent vs. 38.4 percent), to be placed on a waiting list (30.9 percent vs. 17.4 percent), and to undergo transplantation (10.3 percent vs. 2.2 percent, P<0.001 for all three comparisons). CONCLUSIONS: Racial disparities in rates of renal transplantation stem from differences in clinical characteristics that affect appropriateness as well as from underuse of transplantation among blacks and overuse among whites. Reducing racial disparities will require efforts to distinguish their specific causes and the development of interventions tailored to address them.

Adult↗

PubMed2000

Spin-label electron spin resonance studies on the mode of anchoring and vertical location of the N-acyl chain in N-acylphosphatidylethanolamines.

Electron spin resonance (ESR) studies have been performed on N-myristoyl dimyristoylphosphatidylethanolamine (N-14-DMPE) membranes using both phosphatidylcholines spin-labeled at different positions in the sn-2 acyl chain and N-acyl phosphatidylethanolamines spin-labeled in the N-acyl chain to characterize the location and mobility of the N-acyl chain in the lipid membranes. Comparison of the positional dependences of the spectral data for the two series of spin-labeled lipids suggests that the N-acyl chain is positioned at approximately the same level as the sn-2 chain of the phosphatidylcholine spin-label. Further, similar conclusions are reached when the ESR spectra of the N-acyl PE spin-labels in dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylethanolamine (DMPE) host matrixes are compared with those of phosphatidylcholine spin-labels in these two lipids. Finally, the chain ordering effect of cholesterol has also been found to be similar for the N-acyl PE spin-label and PC spin-labels, when the host matrix is either DMPC and cholesterol or N-14-DMPE and cholesterol at a 6:4 mole ratio. In both cases, the gel-to-liquid crystalline phase transition is completely abolished but cholesterol perturbs the gel-phase mobility of N-14-DMPE more readily than that of DMPC. These results demonstrate that the long N-acyl chains are anchored firmly in the hydrophobic interior of the membrane, in an orientation that is parallel to that of the O-acyl chains, and are located at nearly the same vertical position as that of the sn-2 acyl chains in the lipid bilayer. There is a high degree of dynamic compatibility between the N-acyl chains and the O-acyl chains of the lipid bilayer core, although bilayers of N-acyl phosphatidylethanolamines possess a more hydrophobic interior than phosphatidylcholine bilayers. These results provide a structural basis for rationalizing the biological properties of NAPEs.

Cholesterol↗

PubMed2000

Comparative dynamics and location of chain spin-labelled sphingomyelin and phosphatidylcholine in dimyristoyl phosphatidylcholine membranes studied by EPR spectroscopy.

The dynamics and environment of sphingomyelin spin-labelled at different positions in the N-acyl chain have been studied in dimyristoyl phosphatidylcholine bilayer membranes by using electron spin resonance spectroscopy. Comparison was made with phosphatidylcholine spin-labelled on the sn-2 acyl chain in the same host membrane. Spin-labelled sphingomyelin was found to mix well with the host phosphatidylcholine lipids in both gel and fluid phase membranes. At 1 mol%, mutual spin-spin interactions are no greater than for spin-labelled phosphatidylcholine. In the fluid membrane phase, the effective chain order parameters and polarity-sensitive isotropic hyperfine coupling constants of spin-labelled sphingomyelin display a similar dependence on the position of labelling to those of spin-labelled phosphatidylcholine. The values of both parameters are, however, generally larger for sphingomyelin than for phosphatidylcholine at equivalent positions of acyl chain labelling. This difference is attributed to the different chain linkage of sphingo- and glycero-lipids, combined with an offset of approximately one C-atom in transbilayer register between the respective N-acyl and O-acyl chains. In the gel phase, differences in chain configuration between sphingomyelin and phosphatidylcholine are indicated by differences in spin label spectral anisotropy between the two lipids, which appears to reverse towards the terminal methyl chain end.

Dimyristoylphosphatidylcholine↗

PubMed2000

Mixed membranes of sphingolipids and glycerolipids as studied by spin-label ESR spectroscopy. A search for domain formation.

The temperature dependences of the ESR spectra from different positional isomers of sphingomyelin and of phosphatidylcholine spin-labeled in their acyl chain have been compared in mixed membranes composed of sphingolipids and glycerolipids. The purpose of the study was to identify the possible formation of sphingolipid-rich in-plane membrane domains. The principal mixtures that were studied contained sphingomyelin and the corresponding glycerolipid phosphatidylcholine, both from egg yolk. Other sphingolipids that were investigated were brain cerebrosides and brain gangliosides, in addition to sphingomyelins from brain and milk. The outer hyperfine splittings in the ESR spectra of sphingomyelin and of phosphatidylcholine spin-labeled on C-5 of the acyl chain were consistent with mixing of the sphingolipid and glycerolipid components, in fluid-phase membranes. In the gel phase of egg sphingomyelin and its mixtures with phosphatidylcholine, the outer hyperfine splittings of sphingomyelin spin-labeled at C-14 of the acyl chain of sphingomyelin are smaller than those of the corresponding sn-2 chain spin-labeled phosphatidylcholine. This is in contrast to the situation with sphingomyelin and phosphatidylcholine spin-labeled at C-5, for which the outer hyperfine splitting is always greater for the spin-labeled sphingomyelin. The behavior of the C-14 spin-labels is attributed to a different geometry of the acyl chain attachments of the sphingolipids and glycerolipids that is consistent with their respective crystal structures. The two-component ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled at C-14 of the acyl chain directly demonstrate a broad two-phase region with coexisting gel and fluid domains in sphingolipid mixtures with phosphatidylcholine. Domain formation in membranes composed of sphingolipids and glycerolipids alone is related primarily to the higher chain-melting transition temperature of the sphingolipid component.

Electron Spin Resonance Spectroscopy↗

PubMed2000

Fatty acid binding sites of serum albumin probed by non-linear spin-label EPR.

A novel form of non-linear EPR spectroscopy, viz. the first harmonic absorption spectrum recorded in phase quadrature with respect to the Zeeman field modulation, is used here to investigate spin-lattice relaxation enhancements of nitroxide spin labels bound to serum albumin that are induced by spin-spin interactions with aqueous paramagnetic ions. The advantage of this EPR method is that it is directly sensitive to spin-lattice relaxation and affected relatively little by other spectral parameters (Livshits et al., J. Magn. Reson. 133 (1998) 79-91). Relaxation enhancements by ferricyanide of bound fatty acids (n-SASL) spin-labelled at different positions, n, in the chain are compared with those of different maleimide spin label derivatives attached at the single free -SH group, as well as with those of the spin labels free in solution. It was found that: (1) the encounter frequency of ferricyanide with 5-SASL and 12-SASL bound to serum albumin is more than two times less than that with 16-SASL; (2) the accessibility of ferricyanide to 16-SASL is comparable to that of the more immobilised covalently bound spin labels; and (3) the absolute values of the encounter frequencies for the bound spin-labelled fatty acids are approximately a factor of ten smaller than for the corresponding free spin labels, but the latter show a dependence on position of labelling that is similar to the bound labels. A kinetic scheme that is consistent with these relative differences involves rapid reversible transitions between an 'open' and 'closed' state, in which interaction with aqueous paramagnetic agents is possible only in the 'open' state. The equilibrium strongly favours the 'closed' state, which is further enhanced at low temperatures.

Binding Sites↗

PubMed2000

Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents.

The mitochondrial protein horse heart cytochrome c was specifically spin-labeled with succinimidyl-2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectively. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled protein was bound to negatively charged phospholipid membranes composed of dioleoylphosphatidylglycerol, and the accessibility of the spin-labeled lysine residues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled in the headgroup region, in the presence of unlabeled protein, identify the position of the spin-labeled lysine residues relative to the phospholipid bilayer surface. We have found that cytochrome c does not penetrate into the membrane interior and that the active side of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.

Animals↗

PubMed2000

Infrared dichroism of twisted beta-sheet barrels. The structure of E. coli outer membrane proteins.

The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis. For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet. Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels. Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees. Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel.

Amides↗

PubMed2000

Nonlinear electron paramagnetic resonance studies of the interaction of cytochrome c oxidase with spin-labeled lipids in gel-phase membranes.

The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains.

Animals↗

PubMed2000

Spin relaxation measurements using first-harmonic out-of-phase absorption EPR signals: rotational motion effects.

A recent survey of nonlinear continuous-wave (CW) EPR methods revealed that the first-harmonic absorption EPR signal, detected 90 degrees out of phase with respect to the Zeeman modulation (V(1)(')-EPR), is the most appropriate for determining spin-lattice relaxation enhancements of spin labels (V. A. Livshits, T. Páli, and D. Marsh, 1998, J. Magn. Reson. 134, 113-123). The sensitivity of such V(1)(')-EPR spectra to molecular rotational motion is investigated here by spectral simulations for nitroxyl spin labels, over the entire range of rotational correlation times. Determination of the effective spin-lattice relaxation times is less dependent on rotational mobility than for other nonlinear CW EPR methods, especially at a Zeeman modulation frequency of 25 kHz which is particularly appropriate for spin labels. This relative insensitivity to molecular motion further enhances the usefulness of the V(1)(')-EPR method. Calibrations of the out-of-phase to in-phase spectral intensity (and amplitude) ratios are given as a function of spin-lattice relaxation time, for the full range of spin-label rotational correlation times. Experimental measurements on spin labels in the slow, intermediate, and fast motional regimes of molecular rotation are used to test and validate the method.

Calibration↗

PubMed2000

Simulation studies of high-field EPR spectra of spin-labeled lipids in membranes.

The high-field (i.e., 94 GHz) membrane EPR spectra of lipids spin labeled in their fatty acid chains have been simulated by using two limiting motional models. The aim was to identify the dynamic origin of the residual (g(xx) - g(yy)) anisotropy observed in the nonaxial EPR spectra of cholesterol-containing membranes. It is concluded that the residual spectral anisotropy arises from in-plane ordering of the lipid chains by cholesterol. The partial averaging of the (g(xx) - g(yy)) anisotropy was best described by restricted axial rotation with a frequency in the region of tau(-1)(R||) approximately 0.5-1 x 10(9) s(-1). Simulations for slower axial rotation of unrestricted amplitude produced less satisfactory fits. In phospholipid membranes not containing cholesterol, the nonaxial anisotropy is completely averaged in the fluid phase and substantially reduced even in the gel phase. The unrestricted axial rotation in the gel phase is of comparable frequency to that of the limited axial rotation in the liquid-ordered phase of membranes containing cholesterol. These results on in-plane ordering by cholesterol in the liquid-ordered phase could be significant for current proposals regarding domain formation in cellular membranes.

Algorithms↗

PubMed2000

Molecular and mesoscopic properties of hydrophilic polymer-grafted phospholipids mixed with phosphatidylcholine in aqueous dispersion: interaction of dipalmitoyl N-poly(ethylene glycol)phosphatidylethanolamine with dipalmitoylphosphatidylcholine studied by spectrophotometry and spin-label electron spin resonance.

Spin-label electron spin resonance (ESR) spectroscopy, together with optical density measurements, has been used to investigate, at both the molecular and supramolecular levels, the interactions of N-poly(ethylene glycol)-phosphatidylethanolamines (PEG-PE) with phosphatidylcholine (PC) in aqueous dispersions. PEG-PEs are micelle-forming hydrophilic polymer-grafted lipids that are used extensively for steric stabilization of PC liposomes to increase their lifetimes in the blood circulation. All lipids had dipalmitoyl (C16:0) chains, and the polymer polar group of the PEG-PE lipids had a mean molecular mass of either 350 or 2000 Da. PC/PEG-PE mixtures were investigated over the entire range of relative compositions. Spin-label ESR was used quantitatively to investigate bilayer-micelle conversion with increasing PEG-PE content by measurements at temperatures for which the bilayer membrane component of the mixture was in the gel phase. Both saturation transfer ESR and optical density measurements were used to obtain information on the dependence of lipid aggregate size on PEG-PE content. It is found that the stable state of lipid aggregation is strongly dependent not only on PEG-PE content but also on the size of the hydrophilic polar group. These biophysical properties may be used for optimized design of sterically stabilized liposomes.

Dimyristoylphosphatidylcholine↗

PubMed2000

Orientation of the infrared transition moments for an alpha-helix.

Appropriate values for the orientation of the amide transition dipoles are essential to the growing use of isotopically edited vibrational spectroscopy generally in structural biology and to infrared dichroism measurements on membrane-associated alpha-helices, in particular. The orientations of the transition moments for the amide vibrations of an alpha-helix have been determined from the ratio of intensities of the A- and E(1)-symmetry modes in the infrared spectra of poly(gamma-methyl-L-glutamate)(x)-co-(gamma-n-octadecyl-L-glutamate)( y) oriented on silicon substrates. Samples possessing a high degree of alignment were used to facilitate band fitting. Consistent results were obtained from both attenuated total reflection and transmission experiments with polarized radiation, yielding values of Theta(I) = 38 degrees, Theta(II) = 73 degrees, and Theta(A) = 29 degrees, relative to the helix axis, for the amide I, amide II, and amide A bands, respectively. The measurements are discussed both in the context of the somewhat divergent older determinations, and in relation to the helix geometry and results on model amide compounds, to resolve current uncertainties in the literature.

Biophysical Phenomena↗

PubMed2000

Derivatised lipids in membranes. Physico-chemical aspects of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and N-acyl ethanolamines.

The physical properties of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and of N-acyl ethanolamines, in aqueous dispersions, are reviewed. Emphasis is placed on the calorimetric (i.e. chain melting) properties, the thermotropic phase behaviour, certain aspects of the structure and dynamics, and the miscibility with other membrane lipids. In the case of N-biotinyl phosphatidylethanolamines, the specific binding of avidin, and in the case of N-acyl ethanolamines, the function of the third chain, is also considered. All of these properties are relevant to the role of these rather unusual lipids in membranes.

Avidin↗