Talmon Arad

DNA protection by stress-induced biocrystallization

The crystalline state is considered to be incompatible with life. However, in living systems exposed to severe environmental assaults, the sequestration of vital macromolecules in intracellular crystalline assemblies may provide an efficient means for protection. Here we report a generic defence strategy found in Escherichia coli, involving co-crystallization of its DNA with the stress-induced protein Dps,. We show that when purified Dps and DNA interact, extremely stable crystals form almost instantaneously, within which DNA is sequestered and effectively protected against varied assaults. Crystalline structures with similar lattice spacings are formed in E. coli in which Dps is slightly over expressed, as well as in starved wild-type bacteria. Hence, DNA–Dps co-crystallization is proposed to represent a binding mode that provides wide-range protection of DNA by sequestration. The rapid induction and large-scale production of Dps in response to stress, as well as the presence of Dps homologues in many distantly related bacteria, indicate that DNA protection by biocrystallization may be crucial and widespread in prokaryotes.

Rotated plywood structure of primary lamellar bone in the rat: Orientations of the collagen fibril arrays

A basic structural motif of lamellar bone is the arrays of parallel collagen fibrils, with successive arrays having different orientations to form a plywood-like structure. Measurements of the angles between adjacent arrays from cryomicrotomed and vitrified thin sections of demineralized rat bone, cut approximately parallel to the lamellar boundary plane, show that most angles are around 30 degrees, although a subset are around 70 degrees. A structural model for collagen organization based on these measurements is proposed in which an individual lamellar unit (thick and thin lamellae together with transition zones) is composed of five arrays of parallel collagen fibrils, each offset by 30 degrees.

Regulated phase transitions of bacterial chromatin: a non‐enzymatic pathway for generic DNA protection

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient‐depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non‐enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long‐term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Three-dimensional structure of ubiquinol: Cytochrome C reductase from Neurospora mitochondria determined by electron microscopy of membrane crystals

Abstract Membrane crystals have been prepared from mitochondrial ubiquinol: cytochrome c reductase by mixing the enzyme-Triton complex with phospholipid-Triton micelles and subsequently removing the Triton. The electron micrographs of the negatively stained crystals diffract to 2·5 nm, with unit cell dimensions of 13·7 nm by 17·4 nm. The enzyme is arranged in a two-sided plane group P 22 1 2 1 , i.e. alternate molecules span the bilayer in an up and down manner. By combining tilted views of the membrane crystals, a low-resolution three-dimensional structure of the enzyme has been calculated. The structure shows that the enzyme is a dimer, the monomers being related by a 2-fold axis running perpendicular to the membrane. The monomeric units of the enzyme are elongated, extending approximately 15 nm across the membrane. The protein is unequally distributed with about 30% of the total mass located in the bilayer, 50% in a section which extends 7 nm from one side of the bilayer and 20% in a section which extends 3 nm from the opposite side of the bilayer. The two monomeric units are in contact only in the membraneous section. This structure is compared with a model of the enzyme which is derived from biochemical properties of the isolated subunits.

Crystal organization in rat bone lamellae

The plate‐shaped crystals of rat bone are arranged in parallel layers that form coherent structures up to the level of individual lamellae. The crystal layers of the thin lamellae are parallel to the lamellar boundary, whereas those of the thicker lamellae are oblique to the boundary. The basic structure of rat bone can be described as ‘rotated plywood’; a structure hitherto unrecognized in either biologic or synthetic materials.

Halogen Bonding: A Supramolecular Entry for Assembling Nanoparticles

Noncovalent interactions play an important role in the engineering of structurally well-defined assemblies. Important supramolecular forces, including hydrogen and halogen bonding, van der Waals interactions, and p–p stacking have been studied in much detail and used for the design of a vast number of synthons capable of forming task-specific structures having a high level of complexity. Halogen bonding (XB) is an interesting noncovalent interaction in which halogens behave as acceptors of electron density. Recent reports show the increasing significance of XB in liquid crystals, solid-state reactivity, nonporous solids, inorganic chemistry, materials science, and biology, 14] to mention just a few. Remarkably, although XB is considered as a world parallel to hydrogen bonding and a useful tool to construct supramolecular complexes and networks, no studies to date have reported control of the formation and structure of large nanoparticle-based assemblies with this specific and directional interaction. XB interactions are kinetically labile but are considered to be relatively strong. Could, then, this intermolecular force be used to drive and engineer the formation of such assemblies? Herein, we demonstrate the supramolecular assembly of gold nanoparticles (AuNPs) mediated by XB interactions. Our strategy is based on a versatile two-step process. In the first step, the AuNPs were functionalized with an XB donor ligand (1) while particles were kept isolated and their dimensions constant (Scheme 1). Large spherical assemblies were obtained by aging of this system (AuNP–1). Treating AuNP–1 with a bifunctional XB acceptor linker (BPEB) resulted in the formation of chainlike structures or large, dense assemblies, depending on the concentration of BPEB. The dimensions of the spherical particles included in these AuNP–1/BPEB assemblies can be controlled by aging of the AuNP–1 species prior to the reaction with BPEB. Thus, the primary time-dependent assembly of AuNP–1 controls the inner structure, whereas the appearance of the overall structures can be engineered by varying the concentration of the linker (BPEB). Control experiments with an isostructural ligand (2) lacking XB donor capabilities and a monofunctional XB acceptor linker (PEB) highlight the importance of XB interactions in the observed assembly processes. Compound 1 was selected to perform a double role: 1) the coordination of the N-oxide moiety to the surface of the AuNPs provides a relatively stable capping layer preventing the rapid, uncontrolled formation of large colloids, and 2) the ArfI moieties allow the system to form larger structures by means of XB (Scheme 1). Fluorinated aromatic compounds akin to compound 1 containing aryl halides readily form cocrystals with pyridine-containing systems such as BPEB. The order of such structures is often dominated by halogen-bonding interactions. Indeed, the crystal structure of compound 1 reveals that both of the ArfI moieties are involved in XB. Gold nanoparticles capped with tetraoctylammonium bromide (AuNP–TOAB) were used as starting material with an average diameter of (5.4 0.4) nm and a typical surface plasmon band (SPB) at lmax 522 nm in toluene (Figure 1a and Figures 1S and 2S in the Supporting Information). Functionalized AuNPs (AuNP–1) were obtained at room temperature through exchange of TOAB with 1 in organic solvents. The formation of the new AuNP–1 particles by coordination of the polar N-oxide moiety of 1 to the gold surface was verified by optical (UV/Vis) spectroscopy in the transmission mode, which reveals dampening and broadening of the SPB (Figure 1b and Figure 3S in the Supporting Information). Such optical behavior has been reported for various ligand exchange processes, including with thiols, amines, and isothiocyanate. The position of the SPB of metal nanoclusters is influenced by the surrounding media, particularly by the nature of the capping layer. The interactions between the ligands and NPs alter the electron density of the entire system, thus directly affecting the absorption of the surface-bound organic moiety as well as the SPB. The formation of AuNP–1 and the subsequent aggregation process was monitored by UV/Vis spectroscopy and transmission electron microscopy (TEM) during a 48 h time period. This UV/Vis data reveals that the dampening of the SPB develops gradually and is accompanied by a small red shift of approximately 7 nm and band broadening (Figure S3 in the Supporting Information). TEM measurements show that the formation of AuNP–1 occurs within two hours, at which time the sample consists of mainly isolated particles having the same dimensions as the starting material (AuNP– TOAB; Figure 2a and Figure 1S in the Supporting Information). The relative fast TOAB/1 exchange process is followed [*] T. Shirman, Prof. M. E. van der Boom Department of Organic Chemistry, Weizmann Institute of Science 76100 Rehovot (Israel) Fax: (+ 972)8-934-4142 E-mail: milko.vanderboom@weizmann.ac.il Homepage: http://www.weizmann.ac.il/oc/vanderboom/

Ultrastructural studies of bones from patients with osteogenesis imperfecta

Bone samples from patients suffering from osteogenesis imperfecta (OI) types I, II, III and IV, as well as normal controls, were studied by scanning (SEM) and transmission electron microscopy (TEM). SEM views of normal bone at low magnification show coherent structure, with regular striations due to a lamellar plywood-like arrangement of the mineralized collagen fibrils. Compact lamellar bone was also found in various OI specimens, but in limited disconnected regions separated by open spaces. Furthermore, some OI, but not normal, bones have regions of loose unconnected fibers and others of apparently abnormally dense mineral deposition. High resolution TEM studies of OI bone fragments have served to elucidate the structures of these different textures. There appears to be a substantial, though reduced, proportion of normal lamellar bone even in quite severe OI. However, the regions of loose fibers are largely unmineralized and probably contain abnormal collagen. Other regions are overmineralized, with generally small unorganized apatite crystals deposited onto fibril surfaces or in separate clusters. These structural abnormalities, together with the paucity of normal bone, may explain the fragility of OI bones.

Growth of mineral crystals in turkey tendon collagen fibers

Bone and several other vertebrate mineralized tissues are formed by the organized growth of crystals of carbonated apatite within a matrix of type 1 collagen fibers. The development of this process in isolated fibrils of young turkey leg tendons has been studied by transmission electron microscopy. Collagen banding, presumably due to ion concentration, precedes the appearance of any crystals. The smallest crystals observed are short needles in bands near the surface of the fibrils. Longer needles, up to the length of the collagen gap regions, were also seen, and, evidently at a later stage, single crystal belts extending partly or wholly through the fibrils. Finally, in mature tendon crystal platelets, seemingly derived from the cracking of belts, extend partly into the collagen overlap zone. In the least mineralized tendon, extrafibrillar mineral-containing vesicles have occasionally been observed adjacent to regions of radiating needle crystal growth in the fibrils, and, more commonly, smaller particles adjacent to bands of very small needles.

Transitional structures in lamellar bone

Scanning electron micrographs of fractured surfaces of mineralized bone show a lamellar structure with alternating smooth and rough regions. These have been interpreted as corresponding to two distinct collagen fibril and mineral crystal orientations in a rotated plywood structure. However, in various bones, there are clear indications of transition zones between lamellae in which the fibrils, as well as the plate‐like crystals, have intermediate orientations. Strong evidence for intermediate collagen fibril orientations comes from vitrified cryo‐sections of demineralized bone. These show zones of fibril segments graded in length between more homogenous regions of fibrils roughly parallel to the specimen section. Evidence for intermediate crystal orientations comes from transmission electron micrographs and electron diffraction patterns of crushed bone fragments. A tentative scheme is presented for an interlamellar transition zone, involving rotation about the collagen fibril axis as well as tilting of this axis parallel to the plane of the interlamellar boundary. Although it may be convenient to think of the structure of lamellar bone as being composed of alternating thick and thin lamellae, it is probably more correct and biologically more relevant to consider one pair of lamellae as the product of a single depositional cycle of varyingly oriented collagen fibrils that subsequently mineralize.

Three-dimensional image reconstruction from ordered arrays of 70S ribosomes

A better understanding of the molecular mechanism of protein biosynthesis still awaits a reliable model for the ribosomal particle. We describe here the application of a diffraction technique, namely three-dimensional image reconstruction from two-dimensional sheets of 70S ribosomes from Bacillus stearothermophilus at 47 A resolution. The three-dimensional model obtained by these studies shows clearly the two subunits, the contact points between them, an empty space large enough to accommodate the components of protein biosynthesis, the location of regions rich in RNA and a possible binding site for mRNA. The tunnel within the 50S particle which may provide the path taken by the nascent polypeptide chain in partially resolved.