Joachim Seelig

Deuterium magnetic resonance: theory and application to lipid membranes

Proton and carbon-13 nmr spectra of unsonicated lipid bilayers and biological membranes are generally dominated by strong proton–proton and proton–carbon dipolar interactions. As a result the spectra contain a large number of overlapping resonances and are rather difficult to analyse. Nevertheless, important information on the structure and dynamic behaviour of lipid systems has been provided by these techniques (Wennerstrom & Lindblom, 1977).

Lipid conformation in model membranes and biological membranes

Protein molecules in solution or in protein crystals are characterized by rather well-defined structures in which α-helical regions, β-pleated sheets, etc., are the key features. Likewise, the double helix of nucleic acids has almost become the trademark of molecular biology as such. By contrast, the structural analysis of lipids has progressed at a relatively slow pace. The early X-ray diffraction studies by V. Luzzati and others firmly established the fact that the lipids in biological membranes are predominantly organized in bilayer structures (Luzzati, 1968). V. Luzzati was also the first to emphasize the liquid-like conformation of the hydrocarbon chains, similar to that of a liquid paraffin, yet with the average orientation of the chains perpendicular to the lipid–water interface. This liquid–crystalline bilayer is generally observed in lipid–water systems at sufficiently high temperature and water content, as well as in intact biological membranes under physiological conditions (Luzzati & Husson, 1962; Luzzati, 1968; Tardieu, Luzzati & Reman, 1973; Engelman, 1971; Shipley, 1973). In combination with thermodynamic and other spectroscopic observations these investigations culminated in the formulation of the fluid mosaic model of biological membranes (cf. Singer, 1971). However, within the limits of this model the exact nature of lipid conformation and dynamics was immaterial, the lipids were simply pictured as circles with two squiggly lines representing the polar head group and the fatty acyl chains, respectively. No attempt was made to incorporate the well-established chemical structure into this picture. Similarly, membrane proteins were visualized as smooth rotational ellipsoids disregarding the possibility that protruding amino acid side-chains and irregularities of the backbone folding may create a rather rugged protein surface.

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).

Volume-selective excitation. A novel approach to topical NMR

Topical nuclear magnetic resonance (TNMR) is a noninvasive and nonhazardous new technique to obtain high-resolution NMR spectra from a restricted region within a living system. It allows the collection of detailed information about molecular structure, concentration, kinetics, and metabolism in vivo. For detailed TNMR studies of a complex three-dimensional sample such as that of an animal, a precise method for volume selection is crucial. To give the rationale for the development of a new technique, the methods which are presently available to select a sensitive volume are compared briefly. In principle, TNMR spectra can be obtained indirectly via chemical-shift imaging (l-6), a method to investigate the distribution of various chemical compounds throughout the sample, although at significantly reduced sensitivity. To optimize sensitivity, as well as to simplify the experiment and data handling, it is advantageous to use a one-dimensional method to measure a TNMR spectrum. A number of different techniques have been described to restrict the size of the sensitive volume to a predetermined region of interest. They alI rely either on focusing the static magnetic field B0 or on localizing the rf field Br . The methods with static focusing of B0 (7, 8) make use of the fact that highresolution NMR spectra can be obtained only in a volume with high B0 homogeneity. Outside this region, the spectral lines are very much broadened and therefore do not contribute much signal. At the present time, the position of the focused Bo is restricted to the center of the magnet system, which then requires the object under investigation to be moved for every new volume element of interest. Dynamic focusing of BO has also been proposed (9, 10). In this, a steady-state free-precession experiment is performed under the influence of slowly varying linear BO gradients, which eliminate signal contributions from volumes with time dependent Bo. Although this approach allows the selective volume to be moved easily, it su8ers from ill defined boundaries of the sensitive volume and corresponding lineshape problems. The most common method to localize the rf field is the use of a surface rf coil (II). As the name implies, its application is normally restricted to the surface of samples, although efforts have been taken to push the sensitive volume deeper inside the sample by means of special pulse sequences (12, 13). The inherent drawbacks of the methods mentioned above provided the impetus to develop volume-selective excitation (VSE) to localize the sensitive volume. VSE, for which a possible pulse and gradient sequence is given in Fig. 1, is actually a

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.

Orientation and flexibility of the choline head group in phosphatidylcholine bilayers

The average orientation and flexibility of the phosphorylcholine group are deduced from deuterium and phosphorus-31 nuclear magnetic resonance measurements of unsonicated phosphatidylcholine bilayers in the liquid crystalline state. The experimental data are consistent with a model in which the polar head group exhibits a restricted flexibility characterized by rapid transitions between two enantiomeric conformations. A completely flexible or a completely rigid head group structure can be excluded. The phosphorylcholine residue is found to be bent at the position of the phosphate group, due to a gauche-gauche conformation of the phosphodiester linkage. The choline dipole is aligned parallel to the plane of the membrane, which is in agreement with X-ray and neutron diffraction studies. The average orientation of the phosphorylcholine group is therfore the same as that of the phosphorylethanolamine head group.

Structural dynamics in phospholipid bilayers from deuterium spin–lattice relaxation time measurements

The quadrupolar spin–lattice (T1) relaxation of deuterium labeled phospholipid bilayers has been investigated at a resonance frequency of 54.4 MHz. T1 measurements are reported for multilamellar dispersions, single bilayer vesicles, and chloroform/methanol solutions of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC), selectively deuterated at ten different positions in each of the fatty acyl chains and at the sn‐3 carbon of the glycerol backbone. At all segment positions investigated, the T1 relaxation times of the multilamellar and vesicle samples of DPPC were found to be similar. The profiles of the spin–lattice relaxation rate (1/T1) as a function of the deuterated chain segment position resemble the previously determined order profiles [A. Seelig and J. Seelig, Biochem. 13, 4839 (1974)]. In particular, the relaxation rates are approximately constant over the first part of the fatty acyl chains (carbon segments C3–C9), then decreasing in the central region of the bilayer. In chloroform/methanol solu...

Interaction of the Protein Transduction Domain of HIV-1 TAT with Heparan Sulfate: Binding Mechanism and Thermodynamic Parameters

The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal titration calorimetry. The thermodynamic parameters are K0 = (6.0 +/- 0.8) x 10(5) M(-1) and kcal/mol for heparin and K0 = (2.5 +/- 0.5) x 10(5) M(-1) and kcal/mol for chondroitin sulfate B at 28 degrees C. The close thermodynamic similarity of the three binding molecules also implies a close structural relationship. The ubiquitous occurrence of glycosaminoglycans on the cell surface together with their tight and rapid interaction with the TAT protein transduction domain makes complex formation a strong candidate as the primary step of protein translocation.

Titration calorimetry of surfactant-membrane partitioning and membrane solubilization.

The interaction of surfactants with membranes has been difficult to monitor since most detergents are small organic molecules without spectroscopic markers. The development of high sensitivity isothermal titration calorimetry (ITC) has changed this situation distinctly. The insertion of a detergent into the bilayer membrane is generally accompanied by a consumption or release of heat which can be measured fast and reliably with modern titration calorimeters. It is possible to determine the full set of thermodynamic parameters, i.e., the partitioning enthalpy, the partitioning isotherm, the partition coefficient, the free energy, and the entropy of transfer. The application of ITC to the following problems is described: (i) measurement of the critical micellar concentration (CMC) of pure detergent solutions; (ii) analysis of surfactant-membrane partitioning equilibria, including asymmetric insertion; and (iii) membrane-surfactant phase diagrams. Finally, the thermodynamic parameters derived for non-ionic detergents are discussed and the affinity for micelle formation is compared with membrane incorporation.

General features of phospholipid conformation in membranes

In contrast to the well-established conformational properties of proteins and nucleic acids no equivalent structural elements are associated with the phospholipid molecules in fluid bilayer membranes. Instead the hydrocarbon chains are usually represented by a confusion of entangled lines symbolizing the disordered nature of the bilayer interior. This is certainly an oversimplification. We have shown previously that distinct conformational constraints are imposed on the fatty acyl chains of bilayers composed of 3-u+ phosphatidyl cholines [ 11. Still it could be argued that these results are unique for the particular class of phospholipids studied. Here we demonstrate that very similar constraints are obtained for zwitterionic 3-snphosphatidylethanolamine and the negatively charged 3-sn-phosphatidylserine. Furthermore, by using an appropriate normalization procedure the results obtained for the different lipids, including those in biological membranes, were found to agree even quantitatively. Deuterium magnetic resonance (*H-NMR) of selectively deuterated lipids has proved to be a sensitive technique to study the hydrocarbon chain ordering in lipid bilayers [ 1,2]. The method has been applied mainly to bilayers containing the phosphocholine head group, i.e. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) [3,4], 1 -palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) [ 5,6], DPPC plus cholesterol [7], 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) [8] and egg-yolk lecithin [9]. In addition, a deuterium order profile has been published for Acholeplasma laidlawii membranes biosynthetically enriched with deuterated palmitate [lo]. In this