Neutron scattering in the plane of membranes: structure of alamethicin pores.

A technique of neutron in-plane scattering for studying the structures of peptide pores in membranes is described. Alamethicin in the inserted state was prepared and undeuterated and deuterated dilauroyl phosphatidylcholine (DLPC) hydrated with D2O or H2O. Neutron in-plane scattering showed a strong dependence on deuteration, clearly indicating that water is a part of the high-order structure of inserted alamethicin. The data are consistent with the simple barrel-stave model originally proposed by Baumann and Mueller. The theoretical curves computed with this model at four different deuteration conditions agree with the data in all cases. Both the diameter of the water pore and the effective outside diameter of the channel are determined accurately. Alamethicin forms pores in a narrow range of size. In a given sample condition, > 70% of the peptide forms pores of n and n +/- 1 monomers. The pore size varies with hydration and with lipid. In DLPC, the pores are made of n = 8-9 monomers, with a water pore approximately 18 A in diameter and with an effective outside diameter of approximately 40 A. In diphytanoyl phosphatidylcholine, the pores are made of n approximately 11 monomers, with a water pore approximately 26 A in diameter, with an effective outside diameter of approximately 50 A.

Intercalation of small hydrophobic molecules in lipid bilayers containing cholesterol.

Partitioning of small hydrophobic molecules into lipid bilayers containing cholesterol has been studied using the 2XC diffractometer at the University of Missouri Research Reactor. Locations of the compounds were determined by Fourier difference methods with data from both deuterated and undeuterated compounds introduced into the bilayers from the vapor phase. Data fitting procedures were developed for determining how well the compounds were localized. The compounds were found to be localized in a narrow region at the center of the hydrophobic layer, between the two halves of the bilayer. The structures are therefore intercalated structures with the long axis of the molecules in the plane of the bilayer.

Antimicrobial peptide pores in membranes detected by neutron in-plane scattering.

Antimicrobial peptides isolated from the host defense systems of animals have been shown to exert their activity directly on the lipid bilayer of cell membranes, but the antimicrobial mechanisms are not clear, due chiefly to the difficulty of discerning the high-order structures formed by these peptides in membranes. Previously we have shown that these peptides insert into the membrane when their concentrations exceed a lipid-dependent critical value. With neutron in-plane scattering we now show that inserted alamethicin creates aqueous pores approximately greater than 18 A in diameter. The density of pores is consistent with the assumption that all of the alamethicin is involved in pore formation. Pores were not detected below the critical concentration. Thus concentration-dependent pore formation appears to be the molecular mechanism of antimicrobial action.

Atomic force microscopy of purple membranes.

The surface structure of purple membranes was imaged using an atomic force probe mounted in a scanning tunnelling microscope. One of the two different membrane surfaces showed protruding, disc-shaped features forming an hexagonal lattice with about 6 nm centre to centre spacing. These are identified as the cytoplasmic surfaces of trimers of bacteriorhodopsin molecules and are correlated with the structural information on bacteriorhodopsin obtained from numerous earlier electron microscope and diffraction studies.

Electron spin resonance of charge carriers in chlorophyll a/water micelles.

Chlorophyll a/water micelles (P740) prepared in hydrocarbon media have been shown by small-angle neutron scattering to consist of hollow cylinders whose surface is formed of a monolayer of chlorophyll crosslinked by water. The micelles can be reversibly oxidized or reduced to generate highly mobile holes or electrons that undergo rapid, one-dimensional transport along the chains of chlorophyll macrocycles comprising the surface of the micelles. Large pi-pi overlap within the chains facilitates the one-dimensional charge transport and is expected to do the same for energy transport. Structural defects in the micelle surface act as boundaries for charge transport, confining the spins to one-dimensional domains of approximately 200 macrocycles. The one-dimensional transport within the limited domains results in motionally narrowed electron spin resonance lines with some residual inhomogeneous broadening. Although the chlorophyll a incorporated in micelles is more easily oxidized than is monomeric chlorophyll a, it is much more resistant to chemical alteration.

The transducin cascade is involved in the light-induced structural changes observed by neutron diffraction on retinal rod outer segments.

Time-resolved neutron diffraction on retinal rod outer segments are performed to reinvestigate the origin of the light-induced structural change observed by Saibil et al. (Saibil, H., M. Chabre, and D. L. Worcester, 1976, Nature (Lond.), 262:266-270). Photoactivating rhodopsin triggers in rods a cascade of GTP-dependent and transducin-mediated reactions controlling cyclic-GMP hydrolysis. Infrared light-scattering studies (Kühn, H., N. Bennett, M. Michel-Villaz, and M. Chabre, 1981, Proc. Natl. Acad. Sci. USA, 78:6873-6877; Vuong, T. M., M. Chabre, and L. Stryer, 1984, Nature (Lond.), 311:659-661) demonstrated the existence of structural changes that correspond to this cascade rather than to rhodopsin photoactivation. We thus look for neutron diffraction changes of similar origins. With 1-min time resolution, intensity changes are observed mainly for orders 2 and 4. The illumination and GTP dependence of these changes indicates an involvement of transducin. Without GTP, they are linear with the amount of photoexcited rhodopsin, saturate at 10% photolysis, and thus correlate well with the light-scattering "binding signal." With GTP, light sensitivity is higher and saturation occurs below 0.5% photolysis, as for the "dissociation signal" of light scattering. In both cases, lattice compressions of 0.2-0.3% are observed. With 4-s time resolution the intensity change with GTP present precedes the lattice compression. The fast intensity change is probably due to the displacement of transducin alpha-subunits away from the disc membrane and the slower lattice shrinkage to an osmotic readjustment of the rod.

Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure.

By use of neutron diffraction, the structural parameters of oriented multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine with deuteriocarbon chains/cholesterol (molar ratio 70:30), multilamellar lipid vesicles composed of pure lipids and lipid/cholesterol mixtures, and crystalline purple membrane patches from Halobacterium halobium have been measured at pressures up to 2 kbar. Pressurization of the oriented 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/cholesterol multilayers results in an in-plane compression with the mean deuteriocarbon chain spacing of 4.44 A obtained under ambient conditions decreasing by 3-7% at 1.9 kbar. The thickness for this bilayer increases by approximately equal to 1.5 A, but the net bilayer volume decreases and the isothermal compressibility is estimated to be in the range (-0.1 to -0.6) X 10(-4)/bar at 19.0 degrees C. The d spacings for multilamellar vesicles composed of lipids in the liquid crystalline state and lipid/cholesterol mixtures increase linearly as a function of pressure, suggesting that these bilayers are also compressed in the membrane plane. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine MLVs in the gel state, the d spacing decreases with pressure. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, the hexagonally packed chains are anisotropically compressed in the bilayer plane, resulting in a pseudohexagonal chain packing at 1.9 kbar. The bilayer compressibility is (-0.4 or -0.5) X 10(-4)/bar depending on whether the chain tilt increases with pressure or terminal methyl groups of apposing lipid monolayers approach each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Small-angle neutron scattering studies of chlorophyll micelles: Models for bacterial antenna chlorophyll.

Micelles of hydrated chlorophyll a (P740), bacteriochlorophyll a (P865), bacteriochlorophyll c (P750), and pheophytin a prepared in organic media have been studied by small-angle neutron scattering to determine their shape, size, and mass per unit length. All of the micelles are hollow cylinders of well-defined size. The P740 and P750 cylinders are essentially monolayers of macrocycles crosslinked by water, probably in an arrangement similar to that of crystals of chlorophyll derivatives. The P865 micelle is more nearly a bilayer of macrocycles. We show that the curvature necessary to form cylinders probably results from intrinsic curvature of the five-coordinated chlorophyll macrocycle. Studies of P740 micelle formation and the disaggregating effects of another nucleophile (pyridine) are described. As the P750 micelles are nearly identical in size and optical spectra to the rod-shaped structures observed in chlorosomes, and the P865 micelles have optical properties very similar to the in vivo properties of the long-wavelength antenna of purple photosynthetic bacteria, we propose that features of the hydrated cylindrical micelles of these chlorophylls provide good models for antenna chlorophyll in photosynthetic bacteria.

Hydrostatic pressure induces hydrocarbon chain interdigitation in single-component phospholipid bilayers.

By use of neutron diffraction for structural analysis, the temperature-pressure phase diagrams of several fully hydrated single-component phospholipid bilayers have been explored up to hydrostatic pressures of 2 kbars. The gel to liquid-crystalline phase transition temperature Tm increases linearly with pressure over a 10(-3)-2 kbar range in accordance with the Clausius-Clapeyron relationship giving dTm/dP values of 23.0 degrees C/kbar for 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 28.0 degrees C/kbar for 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The so-called pretransition was not observed in the isothermal pressure experiments, suggesting that no appreciable volume change occurs at this transition. These results are in good agreement with those reported using other techniques. In addition, at pressures higher than the isothermal liquid-crystalline to gel transition pressure, a new pressure-induced phase transition was observed for DPPC and DSPC in which the hydrocarbon chains from apposing monolayers become interdigitated with the chains occupying a cross-sectional area approximately equal to 5% less than in the gel phase. The temperature-pressure phase diagrams show the gel-interdigitated phase boundaries to be highly curved and the minimum pressure at which interdigitation occurs to decrease with increasing hydrocarbon chain length.

Structure of the cholesteryl ester core of human plasma low density lipoproteins: selective deuteration and neutron small-angle scattering.

The structural arrangement of cholesteryl esters in human plasma low density lipoproteins (LDL) has been studied by selective deuteration and neutron small-angle scattering. LDL were labeled by in vitro exchange with two different kinds of deuterated cholesteryl esters, one labeled in the fatty acyl chain (cholesteryl myristate-d27) and the other in the branched side chain of cholesterol (cholesteryl-25,26,27-d7 oleate). Neutron scattering data from deuterated and protonated LDL were compared to identify the locations of the fatty acyl and cholesterol side chain moieties. Below the thermotropic transition, radii of gyration of 60 A and 70 A were obtained for these two domains, respectively, indicating that the cholesteryl nuclei are situated more distantly from the center than the fatty acyl chains. At 37 degrees C, above the thermotropic transition of the cholesteryl esters in LDL, both parts have similar radii of gyration of approximately 56 A. This information is used in a discussion of possible structural models for the apolar lipid core of LDL.

Retinal location in purple membrane of Halobacterium halobium: a neutron diffraction study of membranes labelled in vivo with deuterated retinal.

Purple membranes were prepared by growing Halobacterium halobium in a medium containing nicotine (which inhibits biosynthesis of retinal) and the oxidation products of fully deuterated beta-carotene. This allowed the in vivo incorporation of deuterated retinal into the membranes. The labelled membranes were crystalline and isomorphous with native membrane as determined by X-ray diffraction, and their optical absorption spectra were very similar. Neutron diffraction data for the two dimensional in-plane lattice from labelled and native membranes were analysed by difference Fourier and direct methods to 8.6 A resolution. The difference Fourier shows the retinal to be located in the centre of the bacteriorhodopsin molecule. The best fit to the data was obtained with the projection of retinal as a 10 A long rod forming an angle of -40 degrees +/- 10 degrees with the x axis centred at x = -0.19 +/- 0.02, y = -0.35 +/- 0.02 in fractional unit cell coordinates. The main peak in the difference Fourier map is at x = -0.17, y = -0.33.

Structural origins of diamagnetic anisotropy in proteins.

Magnetic anisotropy in proteins and polypeptides can be attributed to the diamagnetic anisotropy of the planar peptide bonds. The alpha helix in particular has large anisotropy due to the axial alignment of the peptide bonds. The regular arrangements of the peptide bonds in beta pleated sheet and collagen structures also produce substantial anisotropy, but less than for alpha helix. The anisotropy permits orientation of small structures of these types in magnetic fields of several kilogauss.

The structure of the chromatin core particle in solution.

The shape and size of the nucleosomal core particle from chromatin has been examined by analysis of neutron and X-ray scattering data from dilute solutions. Calculations of scattering for many different models have been made and only one model was able to account for both the X-ray and neutron profiles. This model is an oblate structure with height about 50A and diameter 110A. The DNA is mainly confined to two annuli located at the top and bottom respectively of the core particle positioned on the outside of a compact protein core which has a height of about 40A and diameter about 73A.