Georg Büldt

Neutron diffraction studies on phosphatidylcholine model membranes : I. Head group conformation

Neutron diffraction experiments on selectively deuterated lipids provide a new method of determining to a segmental resolution the mean conformation of a lipid molecule as projected along the bilayer normal, despite the high amount of disorder that exists in these bilayers. In addition, a time-averaged picture of the extent of the positional fluctuations of the individual segments in this direction can be given. This is demonstrated for a multilamellar system of bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In this paper the head group region of the molecule is examined and this carries the zwitterionic phosphocholine group that determines the electrostatic interaction in the bilayer. Samples deuterated at four different positions in the head group region were measured as oriented samples at 6% (ww) water content at 20 °C (Lβ′ phase) and at 10% (ww) at 70 °C (Lα phase) and as unsonicated dispersions with 25% (ww) water at 28 °C (Lβ′ phase) and 50 °C (Lα phase). From the oriented samples, reflections up to ten orders, and from the powder type samples only four orders, were collected. The derived structure factors for the deuterated segments were fitted assuming a Gaussian distribution of the segments along the bilayer normal. The mean label position was determined for each label under different conditions of water content and temperature with a precision of better than ± 1 angstrom in most cases. The data clearly show that the average orientation of the zwitterionic phosphocholine group is almost parallel to the membrane surface in the gel state (Lβ′) as well as in the liquid crystalline state (Lα). It is interesting to note that in a recent dielectric investigation on this multilamellar system at 25% (ww) water content the same mean orientation of the dipole was found (Shepherd & Buldt, 1978).

Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy

Purple membranes adsorbed to mica were imaged in buffer solution using the atomic force microscope. The hexagonal diffraction patterns of topographs from the cytoplasmic and the extracellular surface showed a resolution of 0.7 and 1.2 nm, respectively. On the cytoplasmic surface, individual bacteriorhodopsin molecules consistently exhibited a distinct substructure. Depending on the pH value of the buffer solution, the height of the purple membranes decreased from 5.6 nm (pH 10.5) to 5.1 nm (pH 4). The results are discussed with respect to the structure determined by cryo-electron microscopy.

Neutron diffraction studies on phosphatidylcholine model membranes: II. Chain conformation and segmental disorder

Abstract In this paper neutron diffraction experiments on 1,2-dipalmitoyl- sn -glycero-3-phosphocholine selectively deuterated in the hydrocarbon chains are reported. The experiments were carried out in the gel phase L β′ and in the liquid crystalline phase L α . The labelled segments were assumed to have a Gaussian distribution in the projection on the bilayer normal and their mean positions were derived with an accuracy of ±1 angstrom unit from a fit to the observed structure factors. The values obtained in the L β′ phase confirm the model with chains in all trans configuration tilted with respect to the bilayer normal by an angle that increases with water content. From samples that were deuterated in both chains separately and studied at low water content it was seen that the chains of the molecule are out of step by as much as 1.5 carbon-carbon bond lengths. A constant width of the label distribution in the projection on the bilayer normal was observed for segments at the beginning and end of the chains. This is an additional indication for the chains being in the all trans state in L β′ phase. In the L α phase, the present experiments show that consecutive segments are well-separated in the profile. The whole chain region is shortened by a factor of ~0.75 compared to the L β′ phase. In contrast to the gel phase, the width of the label distribution is not constant over the entire region, but is found to be increased by more than a factor of two at the end of the chains. This complements the picture derived by deuterium magnetic resonance experiments, where order parameters and correlation times of segmental motions along the chains, which essentially determine the orientational disorder and angular fluctuations of the segments, were obtained.

Zwitterionic dipoles as a dielectric probe for investigating head group mobility in phospholipid membranes

For phospholipid membranes with zwitterionic head groups, the dipole can be considered as a specific label for tracing the changes in the dynamic behaviour of this region of the bilayer in its various phases. Measurements of the dielectric properties of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the frequency range 1--50 MHz show a dispersion which is attributed to the motion of the phosphocholine dipoles in the plane of the bilayers. When the temperature is varied, both the permittivity and loss factor increase sharply at the pretransition (35 degrees C) and the main transition (42 degress C). The relaxation time and amplitude were also determined for this dispersion and these further reflect the structural changes occurring with temperature. The relaxation times varied between 4 ns at 30 degrees C and 2.3 ns at 50 degrees C. Due to steric hindrances a restriction in the angle of head group rotation occurs at lower temperatures but is greatly reduced above the main transition.

The headgroup conformation of phospholipids in membranes.

An important feature which determines the physical state of biological membranes is the great variety of individual lipids occurring in different amounts in the composition of the lipid matrices. One may assume that by this means nature has created conditions which optimize the function of the individual highly specialized protein systems in the membranes of different cells and organelles. Present membrane research is far away from presenting an unambiguous proof for this suggestion. Nevertheless for several reconstituted membrane systems, where one integral membrane protein was embedded in one or more kinds of lipids, it was demonstrated that changes in the spectrum of the hydrocarbon chain conformations and their rates of segmental motions (which determine the fluidity of the bilayer) induces variations in the distribution of the proteins in the plane of the membrane and in their function. Large changes in these properties occur for most phospholipids in bilayers during a first-order phase transition between a so-called gel state and a liquid crystalline state. One clear example for the influence of the lipid state on a protein distribution in the bilayer was given by Kleemann and McConnell (1976) in the reconstitution of Ca-ATPase in bilayers of dimyristoyllecithin. Electron micrographs showed the proteins aggregated in the gel state and more randomly distributed in the liquid crystalline state. Considering lipid influence on membrane protein function, the work of Overath, Schairer and Stoffel (1970) gives an example of the way in which the lipid state influences sugar transport through membranes of Escherichia coli which were enriched in some lipid species. These examples illustrate the influence of the chain conformation on membrane proteins.

Immuno-atomic force microscopy of purple membrane.

The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of purple membrane while imaging in buffer solution. At forces > or = 0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic purple membrane surface was revealed. When the stylus was retracted, the scanned membrane area was relabeled with antibodies within 10 min. The extracellular surface of purple membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immuno-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the purple membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.

The influence of cholesterol on head group mobility in phospholipid membranes

The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0--70 degrees C for various mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency f2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42 degrees C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42 degrees C no sudden changes in f2 are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42 degrees C, however, a decrease in f2 at cholesterol contents up to 20--30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.

Conformational differences between sn-3-phospholipids and sn-2-phospholipids: A neutron and X-ray diffraction investigation☆

Abstract Extensive work has been reported on the conformation in membranes of sn-3-phosphatidylcholines, -ethanolamines, -glycerols and -serines (sn-3-phospholipids), where the headgroup is linked to the third carbon atom in the glycerol backbone. One important feature common to all these naturally occurring phospholipids is that the glycerol moiety is oriented almost perpendicular to the membrane surface, with the sn-1 chain continuing in this direction, whereas the sn-2 chain starts first in a direction parallel to the layer and then bends sharply at the second carbon atom. We present here neutron diffraction results on 1,3-dipalmitoyl-glycero-2-phosphocholine (1,3-DPPC) in which the headgroup is attached to the second carbon atom in the middle of the glycerol part and the two other adjacent carbon atoms are linked to the paraffin chains. Two 1,3-DPPC samples. 2H-labelled at different positions, were studied. One sample was deuterated at the first methylene segment in each fatty acyl chain, and the other at the first segment in one chain and at the second segment in the other chain. The Patterson maps as well as the neutron density profiles show that both fatty acyl chains in 1,3-DPPC have the same conformation introduced by this symmetric chemical bond pattern. It is concluded from this that the C(1)C(3) vector of the glycerol backbone part is oriented parallel to the bilayer surface and the 2H nuclear magnetic resonance signals indicate that both chains have a conformation similar to that observed for the bent chain in sn-3-phospholipids. These findings indirectly confirm the idea that an intramolecular energy minimum, together with the packing geometry of the lipids in the membrane, produce the characteristic conformation around the glycerol backbone as is found for all naturally occurring phospholipids that have been studied so far.

A new approach for using labels in small-angle scattering experiments.

The molecular information which can be obtained from small-angle scattering (SAS) experiments in comparison to crystallography is drastically reduced for two main reasons. (i) The random rotational motions of the molecules in solution eleminate direct access to the orientational information of the structure [ 11. (ii) In SAS experiments there is no amplification of the scattered intensity by a lattice factor as in the case of crystals, therefore resolution is very low. Due to these limitations a unique solution of a structure can normally not be given on the basis of SAS experiments alone. The introduction of parameters characterizing the macromolecule was therefore of great advantage in the development of SAS [2-61. The radius of gyration introduced in [2] is a famous example. Contrast variation was another successful method widely used in neutron small-angle scattering experiments [7,8]. By this procedure the scattering curve can be split up into so-called basic scattering functions which give useful information about different aspects of the structure. The triangulation method is another approach used for macromolecular assemblies, which can be reconstituted from their subunits. The relative positions of the various units is found by determining the distances between pairs of subunits synthetically unhanced in their scattering power [9-l l]. Comprehensive reviews on SAS have appeared in [ 12-141. This paper demonstrates a simple procedure by which additional information can be obtained from the small-angle scattering curve of a molecule which contains a small label of enhanced scattering power. Under these conditions the distance distribution function between the label and each segment in the macromolecule can be determined, which gives amore direct

Specific deuteration and membrane structures.

Diffraction methods have provided a firm time-averaged picture of lipid bilayers and membranes. The basic characteristics of lipid bilayer phases have been obtained, mainly by x-ray diffraction, and some overall mean conformational properties of lipids have been determined (6, 10, 11, 13, 19, 20). As outlined by Tardieu et al. (20), one remarkable feature of lipids is their ability to combine in one phase a periodically ordered long-range organization in one, two, or three dimensions and a highly disordered short-range conformation. This property seems closely related to the function of the lipid matrix in natural membranes. In particular, because of this short-range disorder, it was not possible for a long time to determine the mean conformation of a lipid in a bilayer at higher resolution. Every segment undergoes large positional fluctuations, so that x-ray profiles can give only the superposition of these overlapping segmental distributions, thus hiding the details of the average lipid conformation.