Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing.

Structural studies of CaATPase from sarcoplasmic reticulum have so far been restricted to low resolution due to the poor order of two-dimensional crystal forms. However, we report that three-dimensional microcrystals of detergent-solubilized CaATPase diffract to 7.2 A in x-ray powder patterns and may therefore provide an opportunity to study CaATPase structure at higher resolutions. In the present study, we have characterized the symmetry and molecular packing of negatively stained crystals by electron microscopy (em). By altering the detergent-to-lipid ratio, different sized crystals were produced, which adhere to an em grid in different orientations. Thus, we obtained micrographs of three different projections and from these determined unit cell dimensions to be 151 X 51 X 158 A and the three-dimensional space group to be C2 with an angle beta very close to 90 degrees; x-ray powder patterns of hydrated, unstained crystals yielded dimensions of 166 X 58 X 164 A. Micrographs from each of two principal projections were averaged to produce two-dimensional density maps. Based on these maps and on the previously determined low-resolution structure of CaATPase, a packing diagram for these three-dimensional crystals is presented and major intermolecular contacts are proposed.     

Polymer chain packing analysis using molecular modeling

This article describes the methodology used in applying molecular modeling to investigate the chain packing of polymers. Models for polyethylene and Uans-polyacetylene were developed in order to study the cell packing energy as a function of the chain setting angles. This approach was found to yield chain setting angle values that corresponded to those determined experimentally by other authors. The limitations of the method are also discussed.     

Localization of adriamycin in model and natural membranes. Influence of lipid molecular packing

The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster ovary cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2-APam, 7-ASte, 12-ASte and anthracene-phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Egg-yolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems. Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0-100 microM, the efficiency of energy transfer was 12-ASte greater than 7-ASte greater than 2-APam, with 100% energy transfer for 12-ASte above a drug concentration of 30 microM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7-ASTe greater than 12-ASte = 2-APam was observed. Measurements of fluorescence quenching using the non-permeant Cu2+ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer. Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains. In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of these cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments. These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.     

Occluded molecular surface: Analysis of protein packing

We describe a novel method to calculate the packing interactions in protein structural models. The method calculates the interatomic occluded surface areas for each atom in the protein model. The identification of, and degree of interaction with, neighboring atoms is accomplished by extending surface normal from a dot surface of each atom to the point of intersection with neighboring atoms. The combined occluded and non-occluded surface areas may be normalized for the amino acid composition of the protein providing a single parameter, the normalized protein surface ratio, which is diagnostic for native-like Structures. Individual residues in the model which are in infrequent occluded surface environments may be identified. The method provides a means to explicitly describe packing densities and packing environments of individual atoms in a protein model. Finally, the method allows estimation of the complementarity between any interacting molecules, for example a ligand binding to a receptor.     

Factors affecting molecular packing in mixed lipid monolayers and bilayers

The nature of the molecular interactions and the factors determining molecular packing in mixed phospholipid/glyceride monolayers and bilayers were investigated by monolayer and nuclear magnetic resonance (NMR) techniques. Force-area curves were obtained at various temperatures for monolayers, at the air-water interface, of synthetic lecithins and a phosphatidyl ethanolamine mixed with di- and triglycerides in different molar ratios. The linewidths of peaks in the high resolution NMR spectra of lecithin/glyceride co-dispersions in excess water at different temperatures were used to obtain information about molecular mobilities.It was found that the molecular packing in mixed lipid monolayers and bilayers is determined by the following factors: (1) Whether lipid chains are above or below their melting point (T_C). (2) The difference between experimental temperature and T_C: the larger the difference, the smaller the effect of one component on the other. (3) The degree of similarity of the chains of the components; this influences the degree of cooperativity of chain motions and the degree of mixing of the components. (4) The nature, orientation, mutual interaction and degree of hydration of the polar groups.It is shown that mean molecular area does not always reflect the state of chain motions in lipid films, because of heterogeneity of motion and structure along the molecules. Cooperativity of motion may reduce steric requirements; other effects which are of particular importance for lecithins are interactions of zwitterions, and the influence of polar group hydration.     

Differences in hydrocarbon chain tilt between hydrated phosphatidylethanolamine and phosphatidylcholine bilayers. A molecular packing model.

Both wide-angle and lamellar x-ray diffraction data are interpreted in terms of a difference in hydrocarbon chain tilt between fully hydrated dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE). Although the hydrocarbon chains of multilayers of DPPC tilt ty approximately 30 degrees relative to the normal to the plane of the bilayer, as previously reported by others, the hydrocarbon chains of DPPE appear to be oriented approximately normal to the plane of the bilayer. It is found that the chain tilt in DPPC bilayers can be reduced by either: (a) adding an n-alkane to the bilayer interiors or (b) adding lanthanum ions to the fluid layers between bilayers. A molecular packing model is presented which accounts for these data. According to this model, DPPC chains tilt because of the size and conformation of the PC polar head group.     

Partially systematic molecular packing in the hexagonal columnar phase of dogfish egg case collagen.

The collagen that forms the egg case of the dogfish Scyliorhinus canicula is stored in bulk in the female nidamental glands. Here the collagen molecules are thought to undergo a series of distinct pH-dependent liquid crystalline aggregation phase changes before assembling into the final arrangement encountered in the mature egg case. One liquid crystalline phase is hexagonal with the centres of two adjacent hexagons about 36 nm apart. We have collected tilt series of the hexagonal phase from plastic sections of the nidamental gland and have produced a three-dimensional reconstruction of the collagen arrangement of this phase. The reconstruction features axial columns of protein density lying regularly on the vertices of hexagonal cells of edge length 21 nm. Each column is connected to three nearest neighbours by irregular sheets of protein, but there appear to be preferred molecular directions at about 40 degrees to 50 degrees to the columns. The reconstruction has been interpreted in terms of known interactions of this collagen in other assemblies.     

Structural reorganization of molecular sheets derived from cellulose II by molecular dynamics simulations

We investigated structural reorganization of two different kinds of molecular sheets derived from the cellulose II crystal using molecular dynamics (MD) simulations, in order to identify the initial structure of the cellulose crystal in the course of its regeneration process from solution. After a one-nanosecond simulation, the molecular sheet formed by van der Waals forces along the () crystal plane did not change its structure in an aqueous environment, while the other one formed by hydrogen bonds along the (1 1 0) crystal plane changed into a van der Waals-associated molecular sheet, such as the former. The two structures that were calculated showed substantial similarities such as the high occupancy of intramolecular hydrogen bonds between O3^H and O5 of over 0.75, few intermolecular hydrogen bonds, and the high occurrence of hydrogen bonding with water. The convergence of the two structures into one denotes that the van der Waals-associated molecular sheet can be the initial structure of the cellulose crystal formed in solution. The main chain conformations were almost the same as those in the cellulose II crystal except for a −16° shift of φ (dihedral angle of O5-C1-O1-C4) and the gauche-gauche conformation of the hydroxymethyl side group appears probably due to its hydrogen bonding with water. These results suggest that the van der Waals-associated molecular sheet becomes stable in an aqueous environment with its hydrophobic inside and hydrophilic periphery. Contrary to this, a benzene environment preferred a hydrogen-bonded molecular sheet, which is expected to be the initial structure formed in benzene.     

Glycation induces expansion of the molecular packing of collagen

Exposure of rat tail tendon to a reducing sugar results in covalent attachment of the sugar to collagen, a process termed glycation, and leads to the formation of stable intermolecular cross-links. We have used X-ray diffraction to study the changes in the crystalline unit cell of rat tail tendon collagen brought about by glycation. Ribose was selected as a model compound for most of the study because its reaction with proteins is faster than that of glucose, and therefore more convenient for laboratory studies, but glucose and glyceraldehyde were used as well. A kinetic model describing the process of glycation by ribose and subsequent cross-link formation has been developed. Glycation resulted in an expansion by more than 12% of the unit cell that describes the three-dimensional structure of rat tail tendon collagen. The expansion was in a direction perpendicular to the axes of the rod-shaped molecules, indicating that the intermolecular spacing of the collagen increased. Thus, the structure of collagen in rat tail tendon is significantly altered by glycation in vitro. The expansion was not isotropic, but was directed parallel to the (120) planes, one of the three major planes of the quasi-hexagonal structure that is densely populated by collagen molecules. It is hypothesized that this expansion is brought about by the formation of one, or at most a few, specific intermolecular cross-links in the overlap zone that act to push the molecules apart. It is likely that similar structural changes in collagenous tissues are caused by glycation in vivo during the natural course of aging, and that these changes are accelerated in chronic hyperglycemia such as that associated with diabetes. Analysis of the structure of glycated rat tail tendon potentially can give us new insight into the detailed molecular structure of collagen.     

Steric packing and molecular geometry. I. Simulation on tetrahedral structures of weak covalent bonding

In the first attempt, the molecular geometries of more than 50 tetrahedral structures of f-block and group IVB transition metal organometallic compounds were simulated based on the uniform packing principle. The results are in good agreement with those obtained by X-ray diffraction. It thus provides clear evidence that steric packing plays the dominant role in structures of weak crystal field stabilization energy (CFSE).     

Plasticization, antiplasticization, and molecular packing in amorphous carbohydrate-glycerol matrices

The molecular packing of amorphous maltodextrin-glycerol matrices is systematically explored by combining positron annihilation lifetime spectroscopy (PALS) with thermodynamic measurements and dilatometry. Maltodextrin-glycerol matrices are equilibrated at a range of water activities between 0 and 0.54 at T = 25 °C to analyze the effect of both water and glycerol on the average molecular hole size and the specific volume of the matrices. In the glassy state, glycerol results in a systematic reduction of the average molecular hole size. In contrast, water interacts with the carbohydrate matrix in a complex way. Thermodynamic clustering theory shows that, at very low water contents the water molecules are well dispersed and are closely associated with the carbohydrate chains. In this regime water acts as an antiplasticizer, whereby it reduces the size of the molecular holes. Conversely, at higher water contents, while still in the glassy state, water acts as a plasticizer by increasing the average hole volume of the carbohydrate matrices. This plasticization-dominated mechanism is likely to be due to the interplay between the ability of water to form hydrogen bonds with the hydroxyl residues on the carbohydrate chains and its mobility, which is significantly decoupled from the bulk mobility of the matrix. Our findings are of key importance for the understanding of the effect of glycerol on the biostabilization performance of these carbohydrate matrices, as it provides a first insight on how molecular packing can relate to the dynamics in such matrices.     

Extrafibrillar proteoglycans osmotically regulate the molecular packing of collagen in cartilage

The molecular packing density of collagen and hence the intrafibrillar water content appears to be regulated in cartilage by the osmotic pressure gradient existing between the extrafibrillar and the intrafibrillar compartments.     

Molecular packing in 1-hexanol-DMPC bilayers studied by molecular dynamics simulation

The structure and molecular packing density of a "mismatched" solute, 1-hexanol, in lipid membranes of dimyristoyl phosphatidylcholine (DMPC) was studied by molecular dynamics simulations. We found that the average location and orientation of the hexanol molecules matched earlier experimental data on comparable systems. The local density or molecular packing in DMPC-hexanol was elucidated through the average Voronoi volumes of all heavy (non-hydrogen) atoms. Analogous analysis was conducted on trajectories from simulations of pure 1-hexanol and pure (hydrated) DMPC bilayers. The results suggested a positive volume change, DeltaV(m), of 4 cm(3) mol(-1) hexanol partitioned at 310 K in good accordance with experimental values. Analysis of the apparent volumes of each component in the pure and mixed states further showed that DeltaV(m) reflects a balance between a substantial increase in the packing density of the alcohol upon partitioning and an even stronger loosening in the packing of the lipid. Furthermore, analysis of Voronoi volumes along the membrane normal identifies a distinctive depth dependence of the changes in molecular packing. The outer (interfacial) part of the lipid acyl chains (up to C8) is stretched by about 4%. Concomitantly, the average lateral area per chain decreases and these two effects compensate so that the overall packing density in the outer region, where the hexanol molecules are located, remains practically constant. The core of the bilayer (C9-C13) is slightly thinned. The average lateral area per chain in this region expands, resulting in a looser packing density. The net effect in the core is a 2-3% decrease in density corresponding to a total volume increase of approximately 14 cm(3) mol(-1) hexanol partitioned.     

On the distribution and packing of RNA and protein ribosomes.

From the analysis of the measured radii of gyration of the RNA (Rg = 6.6 +/- 0.3 nm) and protein (Rg = 10.2 +/- 0.5 nm) components of the 50-S subparticle of Escherichia coli ribosomes it is concluded that proteins containing a large amount of hydrodynamically bound water are located on the periphery of the tightly packed RNA. We found that the common features of the measured X-ray scattering curves of the E. coli 70-S ribosome, its 30-S and 50-S subparticles and wheat 80-S ribosomes in the region of scattering angles corresponding to scattering vectors mu from 1 to 5 nm-1 reflect features of the RNA compact packing. A hypothesis is proposed that the compact packing of RNA helices in the range of Bragg distances of 4.5--2.0 nm is a general structural feature of all ribosomal particles.     

Between the sheets: a molecular sieve makes myelin membranes

Myelin is a lipid-rich, spiraled membrane structure that allows for rapid propagation of action potentials through axons. In this issue, Aggarwal et?al. (2011) present evidence that myelin basic protein, essential for myelination by oligodendrocytes, regulates the biosynthesis of myelin membranes by restricting diffusion of membrane-bound proteins into compact myelin.     

Nucleosome packing in interphase chromatin.

Higher-order chromatin fibers (200--300 A in diameter) are reproducibly released from nuclei after lysis in the absence of formalin and/or detergent. Electron microscope analysis of these fibers shows that they are composed of a continuous array of closely apposed nucleosomes which display several distinct packing patterns. Analysis of the organization of nucleosomes within these arrays and their distribution along long stretches of chromatin suggest that the basic 100-A chromatin fiber is not packed into discrete superbeads and is not folded into a uniform solenoid within the native 250-A fiber. Furthermore, because similar higher-order fibers have been visualized in metaphase chromosomes, the existence of this fiber class appears to be independent of the degree of in vivo chromatin condensation.     

Heavy riboflavin synthase from Bacillus subtilis. Particle dimensions, crystal packing and molecular symmetry

Heavy riboflavin synthase from Bacillus subtilis is an enzyme complex consisting of approximately three alpha-subunits (Mr 23.5 X 10(3)) and 60 beta-subunits (Mr 16 X 10(3)). The enzyme has been crystallized from phosphate buffer in a hexagonal crystal modification that belongs to space group P6(3)22. The asymmetric unit of the crystal cell contains ten beta-subunits. The structure of this unusual 10(6) Mr protein has been studied by small-angle X-ray scattering, electron microscopy of three-dimensional crystals, and crystallographic methods. The scattering curves can be interpreted in terms of a hollow sphere model with a ratio of inner and outer radius of 0.3:1. A diameter of 168 A was estimated from the scattering curves, in close agreement with electron microscopic studies. An aggregate with the stoichiometry beta 60, which was obtained by ligand-driven reaggregation of isolated beta-subunits, showed similar shape and dimensions, but a larger value for the ratio Ri/Ra. Electron micrographs of freeze-etched enzyme crystals showed approximately spherical molecules, which were arranged in hexagonal layers. The lattice constants found from the micrographs are in good agreement with the values derived from X-ray diffraction data. Rotation function calculations in Patterson space showed a set of peaks for 2-fold, 3-fold and 5-fold local rotation axes, accurately consistent with icosahedral symmetry and with the particle orientation A shown in the Appendix. The crystal packing can be described as follows: enzyme particles with icosahedral symmetry (point group 532) are located at points 32 of the hexagonal cell, corresponding to positions (0, 0, 0) and (0, 0, 1/2) on the 6-fold screw axes. From the data reported, it may be concluded that the enzyme structure can be described as an icosahedral capsid of 60 beta-subunits with the triangulation number T = 1. The alpha-subunits are located in the central core space of the capsid, but their spatial orientation is incompletely understood.