A dynamic x-ray diffraction study of anesthesia action. Thickening of the myelin membrane by n-pentane.

The structural changes induced in the myelin sheath by n-pentane nerve impulse blockage were studied by small-angle X-ray diffraction using a linear position-sensitive detector. The results show that the thickness of the myelin period lattice increases from 170 to 180 A during n-pentane treatment.     

Cross-bridge conformation as revealed by x-ray diffraction studies on insect flight muscles with ATP analogues.

The effects of three ATP analogues, alpha,beta-methylene-ATP [ATP(alpha,beta-CH1)], adenosine 5'-0-(3-thiotrophosphate) [ATP(gamma-S)], and beta,gamma-amino-ATP [ATP(beta,gamma-NH)] at various concentrations and temperatures on the X-ray fiber diagrams of glycerinated flight muscles from a water bug (Lethocerus maximus) have been investigated. It is shown that the "relaxed" state can be obtained with all three analogues at high concentrations, the result being particularly clear with ATP(gamma-S). It is inferred that the binding of an ATP-like molecule suffices to produce the relaxed state. At low concentrations ATP(beta,gamma-NH) produces state intermediate between rigor and relaxed which is not simply a mixture of the two. The possible nature of the intermediate is discussed.     

Structural states of myelin observed by x-ray diffraction and freeze- fracture electron microscopy

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.     

Low-angle x-ray diffraction of the erythrocyte membrane

Using low-angle X-ray diffraction technique it has been shown that preparing procedures can induce the phase separation in red blood cell membranes in the course of which proteins are removed from lipid bilayers. The latter form rather extensive regions and the diffraction pattern of asymmetric type changes to a symmetric one.     

On phasing the small-angle x-ray diffraction pattern from nerve myelin.

Using a method they developed, Stamatoff and Krimm (1976) have phased swelling data from nerve myelin. Although most phases agree with those I determined previously, there are a few differences. In this letter the two different phasings, theirs and my own, are used to compute the corresponding electron-density profiles, which are then closely compared. For both phasings, small differences are seen in the membrane profile at different degrees of swelling. The explanation that these differences are due simply to errors in measuring intensity is shown to be quite improbable; thus the differences indicate a real change in the profile. It follows that the assumption of a constant membrane profile appears to be invalid in the case of myelin swelling. The differences therefore are assumed to indicate a real change in the profile. It is shown that this change can be attributed consistently to interdigitation of protein molecules at the surfaces of neighboring membranes, while the membrane structure itself remains unchanged. In this case, valid phases still can be determined by swelling, but the phases determined by Stamatoff and Krimm are not valid.     

Interdigitated structure of phospholipid-alcohol systems studied by x-ray diffraction.

In the interdigitated structure of phosphatidylcholine/alcohol systems, the one-dimensional electron density profile in the direction normal to the membrane surface is generated from the x-ray diffraction pattern. The membrane thickness for these systems is expressed by the sum of the hydrocarbon chain lengths of phosphatidylcholine and alcohol molecules. For this study, various sets of phosphatidylcholines and 1-alcohols were used; a phosphatidylcholine has a carbon number from 14 to 18 in a hydrocarbon chain, and an alcohol has a carbon number from 1 (methanol) to 4 (1-butanol). Based upon the results, we propose a model for the interdigitated structure in which 1) two alcohol molecules occupy a volume whose surface is surrounded interstitially by the headgroups of phosphatidylcholine molecules, and 2) the methyl ends of both hydrocarbon chains in alcohol and phosphatidylcholine molecules face each other at the bottom of the volume.     

Repetitive propagation of action potentials destabilizes the structure of the myelin sheath. A dynamic x-ray diffraction study

Time courses of myelin lattice swelling in toad sciatic nerves preexposed to different treatments were determined by x-ray diffraction using a one-dimensional position-sensitive detector. In the nerves supramaximally stimulated for 1 h at 200 Hz, the subsequent process of myelin swelling occurred 45.0 +/- 7.3 min (n = 24) sooner than in resting controls. Sciatic nerves incubated for 1 h in a Ringer's solution deprived of divalent cations (Ca++ and Mg++) exhibited a kinetics of swelling similar to that shown by the stimulated nerves, that is, 52.5 +/- 14.2 min (n = 6) sooner than controls preincubated for the same time in normal Ringer's solution (with divalent cations). The fact that both pretreatments supramaximal stimulation and removal of divalent cations from the perfusion solution produced a similar effect; namely, a decrease of the myelin lattice stability against swelling in distilled water, suggests that the repetitive propagation of action potentials could modify the ionic composition at either the intraperiod channel or the paranodal axoglial junction complexes.     

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.     

Time-resolved x-ray diffraction study of photostimulated purple membrane.

A nanosecond resolution laser-driven x-ray source has been used to perform a time-resolved, x-ray diffraction study of the purple membrane of the Halobacterium halobium. Alterations in diffraction patterns have been observed 1 ms after photostimulation, and are interpreted to show disorder of bacteriorhodopsin packing in the plane of the membrane with little bacteriorhodopsin structural change.     

Sequence of rubredoxin by x-ray diffraction

A tentative amino acid sequence has been determined for rubredoxin from Clostridium pasteurianum by examining the shapes of the side chains in an electron density map at 2 Å resolution. Superpositions of the appropiate portions of this map and “ball and stick” representations of the amino acids assigned are presented as evidence in support of our identifications. Discrepancies exist between the sequence shown here and that determined by chemical methods. The sequence deduced from the 2Å resolution map, however, represents our best estimate in the absence of chemical data and before refinement of the structure. We emphasize the results at 2 Å resolution because, until recently, the 2 Å resolution map was usually the final result of an X-ray structure determination. It is instructive to show the level of reliability that can be expected at that stage and to illustrate the kinds of mistakes that are likely to occur when making identifications from electrondensity maps.     

Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering

Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alpha beta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alpha beta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of alpha subunits in the (alpha beta)4 holoenzyme is similar to that in the tetrameric alpha 4 complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha 4 was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alpha beta holoenzyme, a putative alpha beta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha 4, but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alpha beta protomers, which is consistent with the available biochemical evidence.     

Structure and location of amiodarone in a membrane bilayer as determined by molecular mechanics and quantitative x-ray diffraction.

Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.     

Fluid bilayer structure determination by the combined use of x-ray and neutron diffraction. II. "Composition-space" refinement method.

This is the second of two papers describing a method for the joint refinement of the structure of fluid bilayers using x-ray and neutron diffraction data. We showed in the first paper (Wiener, M. C., and S. H. White. 1990. Biophys. J. 59:162-173) that fluid bilayers generally consist of a nearly perfect lattice of thermally disordered unit cells and that the canonical resolution d/hmax is a measure of the widths of quasimolecular components represented by simple Gaussian functions. The thermal disorder makes possible a "composition space" representation in which the quasimolecular Gaussian distributions describe the number or probability of occupancy per unit length across the width of the bilayer of each component. This representation permits the joint refinement of neutron and x-ray lamellar diffraction data by means of a single quasimolecular structure that is fit simultaneously to both diffraction data sets. Scaling of each component by the appropriate neutron or x-ray scattering length maps the composition space profile to the appropriate scattering length space for comparison to experimental data. Other extensive properties, such as mass, can also be obtained by an appropriate scaling of the refined composition space structure. Based upon simple bilayer models involving crystal and liquid crystal structural information, we estimate that a fluid bilayer with hmax observed diffraction orders will be accurately represented by a structure with approximately hmax quasimolecular components. Strategies for assignment of quasimolecular components are demonstrated through detailed parsing of a phospholipid molecule based upon the one-dimensional projection of the crystal structure of dimyristoylphosphatidylcholine. Finally, we discuss in detail the number of experimental variables required for the composition space joint refinement. We find fluid bilayer structures to be marginally determined by the experimental data. The analysis of errors, which takes on particular importance under these circumstances, is also discussed.     

Structure of the outer mitochondrial membrane analysis of x-ray diffraction from the plant membrane

X-ray diffraction from centrifugally oriented specimens of plant outer mitochondrial membranes suggests that these membranes contain prominent in-plane subunits. The short lamellar repeat which these specimens display (as low as 5.1 nm) points to a predominantly internal localization of the protein components of these membranes. The simplest model for the putative in-plane subunit consistent with autocorrelation analysis of the normal-incidence diffraction data consists of two concentric rings of electron density with diameters of (approx.) 2 and 4 nm. These rings could represent the planar projections of concentric cylindrical shells, aligned normal to the membrane surface.