O. Hayes Griffith

Lipid spin labels in lecithin multilayers. A study of motion along fatty acid chains

Oriented multilayers may be prepared by drying phospholipid dispersions on a glass slide. They provide a convenient model with which to examine motion and orientation in lipid bilayer structures. The lecithin multilayers were examined using a series of four spin labels, consisting of nitroxide free radicals bonded to the 5, 7, 12 or 16 position of stearic acid. Accurate electron spin resonance tensor elements needed to interpret the data were obtained using the nitroxide 2-doxylpropane (the 4′, 4′-dimethyloxazolidine-N-oxyl derivative of acetone) oriented in host single crystals of 2,2,4,4-tetramethylcyclobutanedione. In the multilayers, all four lipid spin labels were found to orient with their long axes perpendicular to the glass slide, but there is a pronounced systematic variation in anisotropy, dependent on the positional isomer used. The anisotropy of the electron spin resonance spectra was measured and tabulated for various degrees of hydration of the lecithin multilayers. Changes in segmental regions were examined by utilizing temperature to change the motion. The splittings can be accounted for in terms of restricted anisotropic motion, and computer simulations based on restricted anisotropic motion and a Gaussian distribution of orientations are in good agreement with the experimental electron spin resonance spectra. The molecular motion increases as the label is moved further from the carboxylend, and for all four spin labels studied, raising the relative humidity increases the molecular motion of the labels in the lecithin multilayers. The effects of hydration are most pronounced when the label is near the carboxyl group of the stearic acid probe. Relative regional differences in motion along the lipid chains tend to be maintained over a wide temperature range.

Identification and extent of fluid bilayer regions in membranous cytochrome oxidase

Abstract Membranous cytochrome oxidase isolated from mitochondria provides a useful model membrane for studies of hydrophobic lipid association with a fairly well characterized functional protein complex. In order to examine the lipid environments, isotropic and oriented samples of cytochrome oxidase of varying phospholipid content were spin labeled with 5-doxylstearic acid (the 4′,4′-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid), and examined by ESR spectroscopy. The isotropic samples exhibit two spectral components. Based on the relative intensity of the two components, the mobility, and the response to hydration, the two components are interpreted as arising from two lipid environments: (1) a layer of lipid bound to the hydrophobic protein surface (boundary lipid) and (2) fluid phospholipid bilayer regions. The extent of the bilayer regions is estimated from integration of the two components of the ESR spectra for samples with different phospholipid/protein ratios. The anisotropy of macroscopically oriented membranous cytochrome oxidase samples containing 0.33 and 0.49 mg phospholipid/mg protein confirms the presence of phospholipd bilayer regions. A spectral analysis reveals a remarkable similarity between the phospholipid bilayer regions in the cytochrome oxidase model membranes and bilayers formed by the isolated lipids, whereas the boundary lipid component has no counterpart in lipid bilayers.

Orientation Dependence of the Electron Spin Resonance Spectrum of Di‐t‐butyl Nitroxide

Comparisons of theoretical calculations and experimental results are reported for the anisotropy of the hyperfine splitting and g value of the di‐t‐butyl nitroxide free radical. Extensive experimental data were collected with a microwave frequency of 9.5 GHz with supplementary data collected at 35 GHz. Several approximations to the Hamiltonian are discussed and compared to the results of the exact solution. Explicit equations for several approximations are given in terms of the principal values of the splitting and g‐value tensors and the orientation of the nitroxide in the external magnetic field. The more accurate approximations should prove useful in the interpretation of nitroxide ESR spectra by decreasing the programming difficulties and the time requirements involved in computer simulation of nitroxide spectra.

Rapid anisotropic motion of spin labels. models for motion averaging of the ESR parameters

Abstract A general approach is developed for averaging of the ESR parameters in the rapid motion limit. It is based on the intermediate-field approximation and is independent of the choice of coordinate systems. Specific models treated include rapid rotation about an arbitrary axis, wobble, and limited amplitude oscillations about one axis of a spin label.

Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids.

The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (betaalpha)(8)-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.

A simplified procedure for lipid phosphorus analysis shows that digestion rates vary with phospholipid structure

A simplified procedure for lipid digestion, well suited for handling a large number of samples, was used to analyze a variety of common phospholipids. This procedure involves digestion of phospholipids in perchloric acid at 130 degrees C with minimal sample manipulation. For all lipids tested, complete destruction, needed for quantitation of phosphate, was achieved after a few hours of digestion under these conditions. Rates of phospholipid destruction, monitored by the spectrophotometric quantitation of released phosphate, varied with lipid structure. Phosphatidic acid (PA), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylinositol (PI) were found to release phosphate faster than phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Although these differences may vary depending on the digestion conditions, they suggest that care should be exercised in lipid phosphate analyses to insure complete digestion.

Fluidity of phospholipid bilayers and membranes after exposure to osmium tetroxide and gluteraldehyde

Abstract The response of fluid bilayer regions to osmium tetroxide and glutaraldehyde fixation was examined in phospholipid multilayers and in nerve bundles from the walking legs of the lobster Homarus americanus . The samples were spinlabeled either with 5-doxylstearic acid (the 4′4′-dimethyloxazolidine- N -ozyl derivative of 5-ketostearic acid) or the maleimide spin label, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl. Osmium tetroxide fixation abolishes the characteristic orientation of the spin-labeled lipid bilayer regions and virtually eliminates motion on the electron spin resonance time scale. Glutaraldehyde treatment reduces the motion of maleimide spin labels covalently attached to proteins. However, in contrast to osmium tetroxide fixation, glutaraldehyde has essentially no effect on the orientation and mobility in the fluid bilayer regions, and hence probably does not restrict directly the potential for translational motion in membrane phospholipid bilayer regions.

THE LIPID‐PROTEIN INTERFACE IN BIOLOGICAL MEMBRANES *

A significant fraction of the lipid in many biological membranes is at the lipid-protein interface. The ESR and NMR data are in basic agreement that there is a dynamic equilibrium between lipid at the interface and the bulk bilayer. The lipid contact with the hydrophobic surfaces of the protein is spatially disordered compared to the bilayer lipids. The spatial disordering on the protein surface leads to the prediction that cooperative chain melting would not occur between lipid tails directly contacting the protein. This is in agreement with most, but not all of the DSC data. While there are some disagreements in the ESR studies, most of the quantitative data support the conclusion that the protein-associated lipid is motionally restricted under physiologically relevant conditions. In general, the NMR data are in agreement that exchange between boundary and bilayer regions is rapid on the NMR time scale at physiological temperatures, although there are some differences in interpretation of the lipid dynamics. From the available data, several kinds of lipid binding sites may be involved. Most of these sites are probably nonspecific, but with some additional sites exhibiting specificity for the chemical properties of the polar head group. The relative binding constants can vary within the boundary layer with several exchange rates applying. Although most of the exchange rates are rapid, perhaps more rapid than specific mechanistic steps in the enzyme reaction, there is a characterizable set of thermodynamic parameters for the boundary and bilayer equilibrium. Although many of the lipid binding sites may have very low relative binding constants, they must be higher than the binding constants for nonspecific protein-protein contacts. One probable function of the boundary is to act as a molecular spacer, preventing indiscriminate protein-protein aggregation in the two-dimensional lipid solvent. Other roles are suggested by the higher relative binding constants of some specific lipids.

The resolution of photoelectron microscopes with UV, X-ray, and synchrotron excitation sources.

The resolution of emission electron microscopes is calculated by determining the intensity distribution in the image. The object is a small disc of uniform brightness centered on the axis. A finite object, as distinct form a point source, provides a non-zero current in the image without the requirement of infinite object brightness and the consequent infinities in the geometrical intensity distribution. The minimum object size, which in turn affects the resolution of the microscope, depends on the minimum current or contrast required in the image. In photoelectron microscopes with UV illumination just above the threshold for emission the predominant aberrations are the chromatic and spherical aberrations of the accelerating field and the spherical aberration of the objective lens. For higher energies, e.g. in the soft X-ray range, the chromatic aberration of the objective lens must also be taken into account, as the aberration coefficients of the accelerating field are greatly reduced. The intensity distributions in the image are calculated first for single energies. The intensity distribution for a beam with a range of energies is obtained by adding a series of single-energy distribution curves weighted according to the energy distribution function. In the presence of spherical aberration the position of the image formed by the electrons depends on the angle of emission. In image planes between the paraxial and marginal planes the combination of spherical aberration and defocus causes the the image spot to have a retrograde type of behavior as the angle of emission increases. The image spot initially moves away from the axis in the azimuth of emission and then returns to the axis and moves away in the opposite azimuth. As a result the intensity in the central portion of the image plane is enhanced. The single-energy intensity distribution curves calculated as a function of depth in the image reveal the existence of a compact, high-intensity image peak in an image plane located between the paraxial and marginal planes. This peak occurs in the plane in which the image spot has a maximum retrograde displacement equal to its radius. The present analysis shows that the resolution in the high-intensity plane is better than in the plane of least confusion, and the effects of aberrations in these two planes are quite different.(ABSTRACT TRUNCATED AT 400 WORDS)

Historical perspective and current trends in emission microscopy, mirror electron microscopy and low-energy electron microscopy: An introduction to the proceedings of the second international symposium and workshop on emission microscopy and related techniques

Emission microscopes and related instruments comprise a specialized class of electron microscopes that have in common an acceleration field in combination with the first stage of imaging (i.e., an immersion objective lens, also called a cathode lens or emission lens). These imaging techniques include photoelectron emission microscopy (PEEM or PEM), electron emission induced by heat, ions, or neutral particles, mirror electron microscopy (MEM), and low-energy electron microscopy (LEEM), among others. In these instruments the specimen is placed on a flat cathode or is the cathode itself. The low-energy electrons that are emitted, reflected, or backscattered from the specimen are first accelerated and then imaged by means of an electron lens system resembling that of a transmission electron microscope. The image is formed in a parallel mode in all of the above instruments, in contrast to the image in scanning electron microscopes, where the information is collected sequentially by scanning the specimen. A brief history and introduction to emission microscopy, MEM, and LEEM is presented as a background for the Proceedings of the Second International Symposium and Workshop on this subject, held in Seattle, Washington, August 16-17, 1990. Current trends in this field gleaned from the presentations at that meeting are discussed.