Spin-label electron spin resonance and Fourier transform infrared spectroscopy for structural/dynamic measurements on ion channels.
Explore the source record for details and available documents.
Amides↗
Biomedical subjects
D Marsh
Publications and source records attributed to D Marsh.
Awake fibreoptic intubation, airway compression and lung collapse in a parturient: anaesthetic and intensive care management.
A 28-year-old primigravida at 35 weeks gestation with acute onset of dyspnoea and stridor due to an intrathoracic neoplasm required semi-urgent caesarean section to allow diagnosis and treatment. Her inability to lie supine precluded regional anaesthesia. She underwent awake fibreoptic oral intubation followed by general anaesthesia. This was complicated by desaturation, high airway pressures, unilateral lung collapse, venous congestion and unexpected blood loss due to an undiagnosed placenta praevia.
Journal Article↗
Interactions at the membrane surface studied by spin label ESR spectroscopy.
A range of different types of interactions at biological membrane surfaces have been studied using various different spin label electron spin resonance (ESR) techniques. These include: (1) the interfacial ionization of local anaesthetics, (2) the binding of peripheral membrane proteins, (3) the membrane insertion of translocation-competent precursor proteins and other components of the protein translocation machinery, (4) the interactions of ganglioside sphingolipids with membrane proteins, and (5) the specific surface recognition of biotinylated phospholipid headgroups by avidin. A description of these illustrates both the capabilities of this biophysical methodology and the functional/technological implications of these interactions and dynamic/thermodynamic processes for cell membranes and their surfaces.
Avidin↗
Effect of precooling on high intensity cycling performance.
OBJECTIVE: To examine the effects of precooling skin and core temperature on a 70 second cycling power test performed in a warm and humid environment (29 degrees C, 80% relative humidity). METHODS: Thirteen male national and international level representative cyclists (mean (SD) age 24.1 (4.1) years; height 181.5 (6.2) cm; weight 75.5 (6.4) kg; maximal oxygen uptake (VO2peak) 66.1 (7.0) ml/kg/min) were tested in random order after either 30 minutes of precooling using cold water immersion or under control conditions (no precooling). Tests were separated by a minimum of two days. The protocol consisted of a 10 minute warm up at 60% of VO2peak followed by three minutes of stretching. This was immediately followed by the 70 second power test which was performed on a standard road bicycle equipped with 172.5 mm powermeter cranks and mounted on a stationary ergometer. RESULTS: Mean power output for the 70 second performance test after precooling was significantly (p<0.005) increased by 3.3 (2.7)% from 581 (57) W to 603 (60) W. Precooling also significantly (p<0.05) decreased core, mean body, and upper and lower body skin temperature; however, by the start of the performance test, lower body skin temperature was no different from control. After precooling, heart rate was also significantly lower than control throughout the warm up (p<0.05). Ratings of perceived exertion were significantly higher than the control condition at the start of the warm up after precooling, but lower than the control condition by the end of the warm up (p<0.05). No differences in blood lactate concentration were detected between conditions. CONCLUSIONS: Precooling improves short term cycling performance, possibly by initiating skin vasoconstriction which may increase blood availability to the working muscles. Future research is required to determine the physiological basis for the ergogenic effects of precooling on high intensity exercise.
Adult↗
Rotational dynamics of spin-labelled surfactant-associated proteins SP-B and SP-C in dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol bilayers.
Pulmonary surfactant proteins SP-B and SP-C have been isolated from porcine lungs and selectively labelled with 2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPO)-isothiocyanate at their N-terminal amine ends, to analyse the mobility of both proteins on the nanosecond time scale using electron spin resonance (ESR) spectroscopy. Reconstitution of the labelled forms of these proteins in bilayers of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) results in much broader and anisotropic ESR spectra, indicating a large restriction in rotational mobility of the protein-attached probe when inserted in membranes. Distinctive differences were found between the ESR spectra of the two polypeptides, that were consistent with intrinsic differences in mode of interaction of SP-B and SP-C with phospholipid bilayers. The mobility of the protein spin probes was sensitive to temperature on the time scale of conventional spin-label ESR. Both proteins, TEMPO-SP-B and TEMPO-SP-C, showed considerable increases in mobility at temperatures above the pretransition of pure DPPC. Finally, the mobility of the spin probes attached to both SP-B and SP-C was more restricted in DPPG than in DPPC bilayers, demonstrating that electrostatic interactions of the positively charged residues at the protein surface influence the rotational dynamics of the proteins in anionic lipid bilayers. Although some residual segmental mobility of the thiourea-linked probes cannot be discounted, the results clearly reflect preferential differences in overall protein dynamics in gel and fluid phases of the two phospholipids that could be important for the biophysical properties of surfactant bilayers and monolayers.
1,2-Dipalmitoylphosphatidylcholine↗
Chain-melting transition temperatures of phospholipids with acylated or alkylated headgroups (N-acyl phosphatidylethanolamines and O-alkyl phosphatidic acids), or with alpha-branched chains.
The biphasic dependence of the chain-melting transition temperature on chainlength, nN, of the headgroup-attached chain of N-acyl phosphatidylethanolamines and of O-alkyl phosphatidic acids is interpreted in terms of a different linear dependence of the transition enthalpy and entropy on nN for long and short chains, respectively. A consistent expression of the form [equation: see text], where DeltanN is the critical chainlength at which the packing mode of the headgroup-attached chains changes, is found to apply systematically to the transition temperatures for both sets of lipids, over the range nN=2-18. This thermodynamic analysis demonstrates that the headgroup-attached chain is located in an environment that differs for long and short chains. Similar considerations apply also to phosphatidylcholines with alpha-branched glycerol-attached chains.
Entropy↗
Structure, dynamics and composition of the lipid-protein interface. Perspectives from spin-labelling.
Implications of the data on lipid-protein interactions involving integral proteins that are obtained from EPR spectroscopy with spin-labelled lipids in membranes are reviewed. The lipid stoichiometry, selectivity and exchange dynamics at the lipid-protein interface can be determined, in addition to information on the configuration and rotational dynamics of the protein-associated lipid chains. These parameters, particularly the stoichiometry and selectivity, are directly related to the intramembranous structure and degree of oligomerisation of the integral protein, and conversely may be used to study the state of assembly of such proteins in the membrane. Insertion of proteins into membranes can be studied by analogous methods. Comparison with the results obtained from integral proteins helps to define the extent of membrane penetration and degree of transmembrane crossing that are relevant to protein translocation mechanisms.
Amino Acid Sequence↗
High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes.
The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.
Anisotropy↗
Cytochrome c-induced increase of motionally restricted lipid in reconstituted cytochrome c oxidase membranes, revealed by spin-label ESR spectroscopy.
Cytochrome c oxidase isolated from beef heart mitochondria was reconstituted in bilayer membranes of the anionic lipid dimyristoylphosphatidylglycerol (DMPG) with varying enzyme/DMPG ratio. Lipid-protein interactions in the reconstituted membrane complexes were studied in the presence and absence of saturating amounts of bound cytochrome c, by both chemical binding assays and spin-label ESR spectroscopy. The ESR spectra from a phosphatidylglycerol probe spin-labeled on C-14 of the sn-2 chain revealed two distinct lipid populations differing in their rotational mobility. The stoichiometry of lipids that were restricted in their rotational motion by direct interaction with the integral protein was 50-60 lipids/cytochrome c oxidase monomer, in the absence of cytochrome c, independent of the total lipid/protein ratio. Cytochrome c alone did not induce a motionally restricted population in the lipid ESR spectra, when bound to bilayers of negatively charged DMPG alone, in the fluid phase (at 36 degreesC). However, the motionally restricted lipid population associated with reconstituted cytochrome c oxidase/DMPG membranes increased on binding cytochrome c, indicating structural/dynamic changes taking place in the membrane. Depending on the DMPG/cytochrome c oxidase ratio, apparent stoichiometries of up to 115 motionally restricted lipid molecules/cytochrome c oxidase monomer were found, when saturating amounts of cytochrome c were bound. Under these conditions, cytochrome c binds to approximately 9 negatively charged DMPG molecules, independent of the cytochrome c oxidase content in the reconstituted system. A likely explanation for these results is that the surface binding of cytochrome c propagates the motional restriction of the lipid chains beyond the first boundary shell of cytochrome c oxidase, possibly creating microscopic in-plane domains.
Animals↗
Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance spectroscopy.
Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using different reconstitution protocols have been estimated from the changes induced in the fluorescence emission spectrum of the single protein tryptophan. All the different reconstituted SP-B-phospholipid preparations studied had similar Kd values for the binding of the protein to the lipids, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein fluorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 A from the center of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as resonance energy transfer from SP-B tryptophan to an acceptor probe located in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on the method of reconstitution. SP-B reconstituted from lipid/protein mixtures in organic solvents is inserted more deeply in PC or DPPC bilayers than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualitative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label electron spin resonance (ESR) spectroscopy, and could represent different functional stages in the surfactant cycle.
Animals↗
Influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers. A 2H NMR and ESR study using designed transmembrane alpha-helical peptides and gramicidin A.
We have investigated the effect of a series of hydrophobic polypeptides (WALP peptides) on the mean hydrophobic thickness of (chain-perdeuterated) phosphatidylcholines (PCs) with different acyl chain length, using 2H NMR and ESR techniques. The WALP peptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked, e.g., FmAW2(LA)nW2AEtn. 2H NMR measurements showed that the shortest peptide with a total length of 16 amino acids (WALP16) causes an increase of 0.6 A in bilayer thickness in di-C12-PC, a smaller increase in di-C14-PC, no effect in di-C16-PC, and a decrease of 0.4 A in di-C18-PC, which was the largest decrease observed in any of the peptide/lipid systems. The longest peptide, WALP19, in di-C12-PC caused the largest increase in thickness of the series (+1.4 A), which decreased again for longer lipids toward di-C18-PC, in which no effect was noticed. WALP17 displayed an influence intermediate between that of WALP16 and WALP19. Altogether, incorporation of the WALP peptides was found to result in small but very systematic changes in bilayer thickness and area per lipid molecule, depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. ESR measurements with spin-labeled lipid probes confirmed this result. Because thickness is expected to be influenced most at the lipids directly adjacent to the peptides, also the maximal adaptation of these first-shell lipids was estimated. The calculation was based on the assumption that there is little or no aggregation of the WALP peptides, as was supported by ESR, and that lipid exchange is rapid on the 2H NMR time scale. It was found that even the maximal possible changes in first-shell lipid length were relatively small and represented only a partial response to mismatch. The synthetic WALP peptides are structurally related to the gramicidin channel, which was therefore used for comparison. In most lipid systems, gramicidin proved to be a stronger perturber of bilayer thickness than WALP19, although its length should approximate that of the shorter WALP16. The effects of gramicidin and WALP peptides on bilayer thickness were evaluated with respect to previous 31P NMR studies on the effects of these peptides on macroscopic lipid phase behavior. Both approaches indicate that, in addition to the effective hydrophobic length, also the physical nature of the peptide surface is a modulator of lipid order.
1,2-Dipalmitoylphosphatidylcholine↗
Selectivity of lipid-protein interactions with trypsinized Na, K-ATPase studied by spin-label EPR.
The selectivity of the lipid-protein interactions in trypsinised Na, K-ATPase membranes from Squalus acanthias has been determined by using EPR spectroscopy with different lipid probes spin-labelled on the 14-C atom of the fatty acid chain. From measurements at low ionic strength and different pH values, the pattern of selectivity is: (stearic acid)->(phosphatidylserine)->(stearic acid)0>(phosphatidylcholine)+/-, where superscripts indicate the formal electrostatic charge on the lipid headgroup. This is in the same order as that determined with native Na,K-ATPase membranes [M. Esmann, D. Marsh, Biochemistry 24 (1985) 3572-3578]. The selectivity for phosphatidylserine is independent of pH, over the range pH 6.0-9. 0, as found also for native membranes. For membranes trypsinised in the presence of Rb+ ions, and in the presence of Na+ (which allows more extensive proteolysis), the relative association constants, Kr, of all lipids are the same as for control membranes, with the exception of ionised (stearic acid)- that shows the highest specificity. Therefore, both the stoichiometry and the principal determinants of the specificity of lipid-protein interaction are preserved on extensive trypsinisation of Na,K-ATPase membranes. This has implications for the location and arrangement of those amino acid side chains that determine the lipid selectivity of the native Na,K-ATPase.
Animals↗
Frequency of codon 1061 and codon 1309 APC mutations in Australian familial adenomatous polyposis patients.
Explore the source record for details and available documents.
Adenomatous Polyposis Coli↗
Relaxation time determinations by progressive saturation EPR: effects of molecular motion and Zeeman modulation for spin labels.
The EPR spectra of nitroxide spin labels have been simulated as a function of microwave field, H1, taking into account both magnetic field modulation and molecular rotation. It is found that the saturation of the second integral, S, of the first harmonic in-phase absorption spectrum is approximated by that predicted for slow-passage conditions, that is, S approximately H1/1 + PH21, in all cases. This result is independent of the degree of inhomogeneous broadening. In general, the fitting parameter, P, depends not only on the T1 and T2 relaxation times, but also on the rate of molecular reorientation and on the modulation frequency. Calibrations for determining the relaxation times are established from the simulations. For a given modulation frequency and molecular reorientation rate, the parameter obtained by fitting the saturation curves is given by 1/P = a + 1/gamma2eT1 . Teff2, where Teff2 is the effective T2. For molecular reorientation frequencies in the range 2 x 10(7)-2 x 10(8) s-1, Teff2 is dominated by the molecular dynamics and is only weakly dependent on the intrinsic T02, allowing a direct estimation of T1. For reorientation frequencies outside this range, the (T1T2) product may be determined from the calibrations. The method is applied to determining relaxation times for spin labels undergoing different rates of rotational reorientation in a variety of environments, including those of biological relevance, and is verified experimentally by the relaxation rate enhancements induced by paramagnetic ions.
Absorption↗
Spin relaxation measurements using first-harmonic out-of-phase absorption EPR signals.
The dependence on spin-lattice (T1) relaxation of the first-harmonic absorption EPR signal (V'1) detected in phase quadrature with the Zeeman modulation has been investigated both theoretically and experimentally for nitroxide spin labels. Spectral simulations were performed by iterative solution of the Bloch equations that contained explicitly both the modulation and microwave magnetic fields (T. Páli, V. A. Livshits, and D. Marsh, 1996, J. Magn. Reson. B 113, 151-159). It was found that, of the various non-linear EPR displays, the first-harmonic out-of-phase V'1-signal, recorded under conditions of partial saturation of the microwave absorption, is particularly favorable for determining spin-lattice relaxation enhancements because of its superior signal intensity and relative insensitivity to spin-spin (T2) relaxation. By varying the Zeeman modulation frequency it is also possible to tune the optimum sensitivity of the V'1-signal to different ranges of the T1-relaxation time. A Zeeman modulation frequency of 25 kHz appears to be particularly suited to spin label applications. Calibrations are given for the dependence on T1-relaxation time of both the amplitude and the second integral of the V'1-signal recorded under standard conditions. Experiments on different spin labels in solution and in membranes demonstrate the practical usable sensitivity of the V'1-signal, even at modulation frequencies of 25 kHz, and these are used to investigate the dependence on microwave field intensity, in comparison with theoretical predictions. The practicable sensitivity to spin-lattice relaxation enhancements is demonstrated experimentally for a spin-labeled membrane system in the presence of paramagnetic ions. The first-harmonic out-of-phase V'1-signal appears to be the non-linear CW EPR method of choice for determining T1-relaxation enhancements in spin-labeled systems.
1,2-Dipalmitoylphosphatidylcholine↗
Nonaxiality in infrared dichroic ratios of polytopic transmembrane proteins.
In polytopic alpha-helical transmembrane proteins, the distribution of amide vibrational transition moments can be nonaxial, if the helix axes are tilted relative to the symmetry axis of the helix bundle. The infrared dichroic ratios from oriented samples then contain nonaxial terms and, in the most general case, require a second-order parameter for the axis of the helix bundle. The extent of nonaxiality depends on the summation over the individual amide transition moments along the helix. Because this is strongly oscillatory, with a 3.6-residue periodicity, complete axial symmetry is not achieved rapidly on progressive summation. Expressions for the contributions of residual nonaxiality to the dichroic ratios are derived. A similar situation arises for oligomers of transmembrane beta-barrel proteins, e.g., the porin trimer. In this case, the extent of nonaxiality depends not only on the number of residues in the beta-barrel, but also on the tilt of the beta-strands relative to the barrel axis and the characteristic dimensions of a beta-sheet, which together determine the axial periodicity. The nonaxial contributions to the dichroic ratios of beta-barrel oligomers are also derived. Estimates are given of the likely size of the nonaxial contributions for the different alpha-helical and beta-sheet systems.
Amides↗
Protein-induced vertical lipid dislocation in a model membrane system: spin-label relaxation studies on avidin-biotinylphosphatidylethanolamine interactions.
The change in vertical location of spin-labeled N-biotinyl phosphatidylethanolamine in fluid-phase dimyristoyl phosphatidylcholine bilayer membranes, on binding avidin to the biotinyl headgroup, has been investigated by progressive saturation electron spin resonance measurements. Spin-labeled phospholipids were present at a concentration of 1 mol%, relative to total membrane lipids. For avidin-bound N-biotinyl phosphatidylethanolamine spin-labeled on the 8 C atom of the sn-2 chain, the relaxation enhancement induced by 30 mM Ni2+ ions confined to the aqueous phase was 2.5 times that induced by saturating molecular oxygen, which is preferentially concentrated in the hydrophobic core of the membrane. For phosphatidylcholine also spin-labeled at the 8 position of the sn-2 chain, this ratio was reversed: the relaxation enhancement by Ni2+ ions was half that induced by molecular oxygen. In the absence of avidin, the enhancement by either relaxant was the same for both spin-labeled phospholipids. For a double-labeled system, in which both N-biotinyl phosphatidylethanolamine and phosphatidylcholine were spin-labeled on the 12 C atom of the sn-2 chain, the relaxation rate in the absence of avidin was greater than that predicted from linear additivity of the corresponding singly labeled systems, because of mutual spin-spin interactions between the two labeled lipid species. On binding of avidin to the N-biotinyl phosphatidylethanolamine, this relaxation enhancement by mutual spin-spin interaction was very much decreased. These results indicate that, on binding of avidin to the lipid headgroup, N-biotinyl phosphatidylethanolamine is lifted vertically within the membrane, relative to the phosphatidylcholine host lipids. The specific binding of avidin to N-biotinyl phosphatidylethanolamine parallels the liftase activity proposed for activator proteins associated with the action of certain gangliosidases.
Animals↗
Recent developments in spin labelling.
Explore the source record for details and available documents.
Biophysical Phenomena↗