Protein-lipid interactions. High-field deuterium and phosphorus nuclear magnetic resonance spectroscopic investigation of the cytochrome oxidase-phospholipid interaction and the effects of cholate

102

2H NMR and EPR spectra have been obtained as a function of temperature and protein concentration from the same samples of beef heart cytochrome c oxidase reconstituted into 1-(16,16,16-trideuteriopalmitoyl)-2-palmitoleoyl-sn-glycero-3-phosphocholine. At all temperatures, the EPR spectra show the characteristic "bound" and "free" components, while the 2H NMR spectra show only a narrow distribution of orientational order parameters. At temperatures near the phase transition of the pure lipid, the dependence of the 2H NMR average orientational order on protein concentration fits a two-stage model in which the phospholipid molecular exchange rapidly between two states tentatively identified as sites either on or off the protein surface. From this model, the 2H NMR spectra yield a value of 0.18 mg of phospholipid per mg of protein as necessary to cover the surface of cytochrome c oxidase, which is the same value as derived from the EPR spectra at -20 degrees C. Both the 2H NMR and EPR spectra vary markedly with temperature. At temperatures well above the phase transition of the pure lipid, the average orientational parameters derived from the 2H NMR spectra are independent of protein concentration and are the same as for the lipid alone. Qualitatively, the EPR spectra show large apparent decreases in the average orientational order with increasing temperature. Analysis of 2H NMR relaxation rates indicates an additional motion in the presence of protein with a correlation time of 10(-6)-10(-7) s. If this new motion is associated with exchange between the two states, a minimum value of 10(6)-10(7) s-1 for the exchange rate is obtained, assuming that the lipids on the protein surface are much more motionally restricted than the rest of the lipid. Such an exchange rate is compatible with the observed differences in 2H NMR and EPR spectra. These results are consistent with short-lived, energetically weak interactions between cytochrome c oxidase and the phospholipids used in this study.

Section: Theorysupporting

confidence: 67%

Dynamical and temperature-dependent effects of lipid-protein interactions. Application of deuterium nuclear magnetic resonance and electron paramagnetic resonance spectroscopy to the same reconstitutions of cytochrome c oxidase

Paddy¹,

Dahlquist²,

Davis³

et al. 1981

102

“…This could be the source of the reduced order in the polar head figure 6: Suggested structure of the hexagonal phase in the lecithin-sodium cholate-water system, showing the continuous hydrocarbon regions of the rodlike aggregates. region observed by Rice et al (1979) through 3IP NMR, by Persson et al (1974) through 2H NMR on the water, and by Lindblom et al (1976a-c) through counterion NMR.…”

Section: Discussionmentioning

confidence: 90%

“…Cholesterol is solubilized with the hydroxyl group at the bilayer surface, and the steroid skeleton seems to orient the acyl chain, possibly by reducing the number of kinks or by reducing the tilt of the chains. Integral membrane proteins appear to affect the acyl chain order to a remarkably small extent even at high weight fractions (Rice et al, 1979;Bloom, 1979;Davis et al, 1980). Although this problem is not yet fully explored, it is clear that these proteins do not mediate an extensive perturbation of the bilayer structure.…”

Section: Discussionmentioning

confidence: 99%

Molecular organization in the liquid-crystalline phases of lecithin-sodium cholate-water systems studied by nuclear magnetic resonance

Ulmius¹,

Lindblom²,

Wennerstroem³

et al. 1982

101

The molecular organization in the hexagonal and lamellar phases of the ternary systems lecithin--sodium cholate--water has been investigated by using a variety of nuclear magnetic resonance techniques. The main findings and conclusions are the following: (i) When calculated on a mole fraction basis, the phase equilibria are insensitive to changes in the alkyl chains of the lecithin. (ii) When incorporated into a lecithin bilayer, cholate exerts a strong perturbation on the lecithin alkyl chain order, giving a large decrease of the order parameters. (iii) This decrease of the order occurs since the average cross-sectional area per alkyl chain increases probably as a result of cholate placing itself flat on the bilayer surface. (iv) The diffusion of lecithin molecules is approximately equally rapid in the lamellar and hexagonal phases. (v) The hexagonal phase is formed by rodlike aggregates with the polar groups at the surface of the rods and with a continuous hydrocarbon core. The rods are not formed by stacking disklike mixed micelles. (vi) With the interpretations of the molecular packing and the phase structures, the observed phase equilibria are in good agreement with current theories of the factors that govern phase behavior in amphiphile--water systems.

“…In contrast, infrared spectra for the hydrophilic F2 fragment are suggestive of a 0 conformation with perhaps spectral contributions from random-coil configurations. The -helical conformation of intact melittin in DMPC multilayer .^Llthough the perturbing effects of lipid-protein associations on the physical and chemical behavior of bilayer systems are well appreciated [see, for example, Seelig & Seelig (1980), Chapman et al (1979), Rice et al (1979), Pink & Chapman (1979), Griffith & Jost (1978), and Papahadjopoulous et al (1975)], relatively little spectroscopic information exists concerning the influence of the lipid environment upon protein conformation. For examination of this problem, a number of the spectroscopic techniques generally used to determine protein conformations in aqueous media have also proven advantageous for clarifying the structural arrangements assumed by proteins either embedded within lipid bilayers or associated with micellar assemblies (Massey et al, 1981;Bósch et al, 1980; Wallace & Blout, 1979; Keniry & Smith, 1979.…”

mentioning

confidence: 99%

Infrared spectroscopic study of the secondary structure of melittin in water, 2-chloroethanol, and phospholipid bilayer dispersions

Lavialle¹,

Adams²,

Levin³

1982

The conformations of melittin, an amphipathic polypeptide consisting of 26 amino acid residues, and its hydrophobic (residues 1--19) and hydrophilic (residues 20--26) fragments were examined in various solvent systems, including H2O, 2H2O, 2-chloroethanol, and 1,2-dimyristoylphosphatidylcholine (DMPC) multilayers, by infrared spectroscopy. Water and 2-chloroethanol were used as reference solvents for characterizing the amide I and II vibrational frequencies of the polypeptide in systems reflecting unordered, beta-structure, or alpha-helical forms. In DMPC bilayer assemblies both melittin and its hydrophobic fragment F1 exhibit alpha-helical conformations. In contrast, infrared spectra for the hydrophilic F2 fragment are suggestive of a beta conformation with perhaps spectral contributions from random-coil configurations. The alpha-helical conformation of intact melittin in DMPC multilayer dispersions remains unchanged as the bilayer passes from the gel to liquid-crystalline state. For melittin-water solutions the infrared spectra monitor changes in population of specific conformations as the temperature is varied. Thus, for melittin concentrations in which tetramers are dominant high temperatures (31 degrees C) favor the alpha-helical form, while low temperatures (8 degrees C) lead to populations of both beta and alpha-helical structures. At lower melittin concentrations for which monomers persist, high temperatures favor an unordered polypeptide form, while low temperatures induce an alpha-helical conformation. Although peak-height intensity ratios AII/AI for the amide I and II regions are difficult to interpret rigorously, values of this parameter for aqueous solutions of melittin suggest a sensitivity to structural changes involving the aggregation properties of the polypeptide.