A-I, the major protein of high density lipoproteins (HDL) and dimyristoyl phosphatidilcholine (DMPC), by the techniques of neutron scattering and electron microscopy. In order to simplify the experiment, we changed contrast by using deuterated (d-DMPC) and hydrogenated (h-DMPC) lipids to form recombinants, and did all of the scattering experiments in D20.

=

50) in

Negative stain electron microscopy of the 40:1 (M:M) recombinants showed discoid structures. The h-DMPC: A-I recombinants measured 99 ± 9 A (standard deviation, n = 150) in diameter and 32 ± 3 Ă (n thickness; the d-DMPC: A-I recombinants measured 98 ± 10 +5A (n

=

50).

A (n

=

150) by 33

In the first neutron scattering experiments, h-DMPC: A-I recombinants were studied at concentrations of 5.75, 11.5, and 23 mg dry weight lipid per ml. Calculation of the radii of gyration showed no concentration

dependence, within experimental error.

Figure 1 shows Guinier plots of low-angle scattering from h-DMPC and d-DMPC recombinants. Radii of gyration calculated from the slopes were 33.5 ± 0.2 Å for h-DMPC and 38.0 ± 0.5 Å for d-DMPC. The normalized square roots of zero-angle intensity were 3.8 and 1.0, respectively. electron microscopic studies indicate that this difference in radii of gyration cannot be explained simply by different diameters of the h- and d-particles.

Data obtained from the Guinier region cannot determine uniquely the structure of particles. However, their consistency with other data may be examined using models based on geometric parameters and the scattering densities of the components. A-I: DMPC discs may be described in terms of

The

three parts, with the scattering density of each assumed homogeneous.
lipids have no readily exchangeable hydrogens, and the average scattering
densities calculated for their non-polar parts are -0.008 x 10-14cm/Å3
(h-DMPC) and 7.3 x 10-14 cm/A3 (d-DMPC). The scattering density of the
polar head group is 0.76 x 10-14 cm/Å3 in each case.

Figure 1.

Guinier plots for apo A-I recombinants containing h-DMPC (curve
1) and d-DMPC (curve 2). Intensity is measured in arbitrary
units, but is scaled for the two samples. h = 4π sine/λ.
Concentrations of recombinant were 24 (curve 1) and 30 mg/ml
(curve 2).

The scattering density for a protein is more difficult to calculate because of uncertainties in hydration and hydrogen exchange. We arrived at the value to be used here by considering the scattering intensity at zero angle. The square root of intensity is proportional only to the sum of scattering lengths of all atoms in the complex, less the sum for displaced solvent molecules; it is not influenced by the distribution of atoms. If the level of hydration of protein is 0.4 g/g, it follows that the volume fraction of protein in the complex is 0.5, that of nonpolar part of the lipid is 0.375, and that of the polar lipid is 0.125. With these values, the scattering density of A-I can be calculated from the zero angle intensity as 5 x 10-14 cm/A3. This corresponds to exchange of 30% of hydrogen atoms for deuterium, in good agreement with a figure of 25% obtained from the amino acid composition.

The larger radius of gyration for d-DMPC (in which the scattering density of the non-polar lipid closely resembles that of D20) clearly indicates that the protein resides in the outer part of the complex. Using the above values for the volume fractions and scattering densities of the components, we searched for model structures in best agreement with the experimental radii of gyration. We considered models based on spherical, spheroidal, and cylindrical geometries with either three separate scattering regions or protein uniformly dispersed throughout the lipid polar head regions. Predicted dimensions for the deuterated and hydrogenous recombinants were in best agreement for a three-component cylindrical model (figure 2) with R = 49 Å and H = 16.3 Å (R1 10.9 Å). Good agreement was not obtained with spherical or mixed protein-lipid models. The best spheroidal model had dimensions similar to those of the optimal

=

34.6 A, H1

=

Figure 2.

A model for apo A-I: DMPC discoid recombinants. Region 1 is occupied by DMPC polar head groups, region 2 by DMPC fatty acyl chains, and region 3 by apo A-I. Detailed discussion in text.

cylindrical one, but the fit was not as good. Thus the AI: DMPC recombinant can be approximated by a disc with diameter of 98 Å and height of 33 Å, in excellent agreement with the dimensions determined by negative stain electron microscopy. In this model the A-I protein forms an outer rim 15 A

Å

thick, the polar DMPC head groups are on the top and bottom and the nonpolar fatty acid chains are buried inside.

High resolution negative stain electron microscopy shows two additional features compatible with the bicycle tire model. First, when the discoid particles orient with their flat faces parallel to the plane of the grid, contrast rims can be seen around the perimeters of the discs. Second, examination of discs oriented edgewise (the usual manner of orientation) reveals two parallel electron-lucencies running the length of the disc rim. These parallel lucencies are each approximately 16 Å thick. As evidence against artifact, both features are seen independently of the grain size or level of focus of the micrographs; however, artifact cannot be ruled out absolutely.

The electron microscopic features and neutron scattering data are compatible with a molecular model consisting of a unilamellar lipid bilayer disc 68 Å in diameter (at the 40:1 DMPC:A-I ratio), whose rim is lined with A-I. The dimensions of the protein rim (R-R1 15 Å; 2H 33 Å) indicate

two A-I molecules per recombinant, consistent with cross-linking studies reported earlier.

A STUDY OF METHYL GROUP LIBRATION IN NITROMETHANE

S. F. Trevino

(Energetic Materials Division, LCWSL, ARRADCOM, Dover, NJ)

and

E. Prince

and

C. R. Hubbard

(Ceramics, Glass, and Solid State
Science Division)

Nitromethane, CH, NO2 freezes at -30°C, forming an orthorhombic structure in space group P212121 with Z=4. Neutron powder diffraction patterns were obtained from a deuterated sample at six temperatures from 4.2K to 125K. Quasielastic neutron scattering showed that the methyl

groups undergo hindered rotation with a potential barrier corresponding to a temperature of 118K. A librational motion would be expected with substantial amplitude even at the lowest temperature. The structure was refined by profile analysis using a constrained model in which the methyl group was allowed to librate around the C-N vector, and also was required to have three-fold symmetry around that vector. At the lowest temperature the libration has a zero point amplitude consistent with the energy levels inferred from neutron spectroscopy. As the temperature is raised, the amplitude increases monotonically in good agreement with theoretical expectations. Even at 125K the hydrogen equilibrium positions are well defined, although the standard deviations are higher at the higher temperatures.