Aquaporin-0 membrane junctions reveal the structure of a closed water pore

The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo. We show here that AQP0 from the lens core, containing some carboxy-terminally cleaved AQP0, forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystallography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating.     

Structural basis of water-specific transport through the AQP1 water channel.

Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients. These channels are believed to be involved in many physiological processes that include renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction. Members of the water channel superfamily have been found in a range of cell types from bacteria to human. In mammals, there are currently 10 families of water channels, referred to as aquaporins (AQP): AQP0-AQP9. Here we report the structure of the aquaporin 1 (AQP1) water channel to 2.2 A resolution. The channel consists of three topological elements, an extracellular and a cytoplasmic vestibule connected by an extended narrow pore or selectivity filter. Within the selectivity filter, four bound waters are localized along three hydrophilic nodes, which punctuate an otherwise extremely hydrophobic pore segment. This unusual combination of a long hydrophobic pore and a minimal number of solute binding sites facilitates rapid water transport. Residues of the constriction region, in particular histidine 182, which is conserved among all known water-specific channels, are critical in establishing water specificity. Our analysis of the AQP1 pore also indicates that the transport of protons through this channel is highly energetically unfavourable.     

Structural determinants of water permeation through aquaporin-1

Human red cell AQP1 is the first functionally defined member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8A resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiology-how membranes can be freely permeable to water but impermeable to protons.     

Rapid gating and anion permeability of an intracellular aquaporin

Aquaporin (AQP) water-channel proteins are freely permeated by water but not by ions or charged solutes. Although mammalian aquaporins were believed to be located in plasma membranes, rat AQP6 is restricted to intracellular vesicles in renal epithelia. Here we show that AQP6 is functionally distinct from other known aquaporins. When expressed in Xenopus laevis oocytes, AQP6 exhibits low basal water permeability; however, when treated with the known water channel inhibitor, Hg2+, the water permeability of AQP6 oocytes rapidly rises up to tenfold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid-secreting alpha-intercalated cells in renal collecting duct. At pH less than 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity, but leaves low pH activation intact. Our results demonstrate unusual biophysical properties of an aquaporin, and indicate that anion-channel function may now be explored in a protein with known structure.     

Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment

Voltage-dependent K+ (Kv) channels repolarize the action potential in neurons and muscle. This type of channel is gated directly by membrane voltage through protein domains known as voltage sensors, which are molecular voltmeters that read the membrane voltage and regulate the pore. Here we describe the structure of a chimaeric voltage-dependent K+ channel, which we call the 'paddle-chimaera channel', in which the voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2. Crystallized in complex with lipids, the complete structure at 2.4 ?ngstr?m resolution reveals the pore and voltage sensors embedded in a membrane-like arrangement of lipid molecules. The detailed structure, which can be compared directly to a large body of functional data, explains charge stabilization within the membrane and suggests a mechanism for voltage-sensor movements and pore gating.     

Structure and gating mechanism of the acetylcholine receptor pore

The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.     

Selective assembly on a surface of supramolecular aggregates with controlled size and shape.

The realization of molecule-based miniature devices with advanced functions requires the development of new and efficient approaches for combining molecular building blocks into desired functional structures, ideally with these structures supported on suitable substrates 1-4.Supramolecular aggregation occurs spontaneously and can lead to controlled structures if selective and directional non-covalent interactions are exploited. But such selective supramolecular assembly has yielded almost exclusively crystals or dissolved structures 5; the self-assembly of absorbed molecules into larger structures 6-8, in contrast, has not yet been directed by controlling selective intermolecular interactions. Here we report the formation of surface-supported supramolecular structures whose size and aggregation pattern are rationally controlled by tuning the non-covalent interactions between individual absorbed molecules.Using low-temperature scanning tunnelling microscopy, we show that substituted porphyrin molecules adsorbed on a gold surface form monomers, trimers, tetramers or extended wire-like structures. We find that each structure corresponds in a predictable fashion to the geometric and chemical nature of the porphyrin substituents that mediate the interactions between individual adsorbed molecules.Our findings suggest that careful placement of functional groups that are able to participate in directed non-covalent interactions will allow the rational design and construction of a wide range of supramolecular architectures absorbed to surfaces.     

Importance of NPA motifs in the expression and function of water channel aquaporin-1

The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and effi-cient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 de-letion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water per-meation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.     

β分子筛中苯分子扩散行为的KMC模拟

采用动力学Monte Carlo（KMC）方法模拟研究了苯分子在β分子筛中的扩散行为,并将计算结果与描述多孔介质中表面扩散的Maxwell－Stefan（M-S）方程进行比较。在模拟过程中,通过引入反映扩散分子间相互作用的参数,计及扩散过程中被吸附分子之间的作用力,得到较常规的KMC方法更为准确的结果,表明：M-S方程可以很好的描述β分子筛中苯分子的扩散,尤其对于M－S扩散系数,由于KMC模拟和M-S方程均考虑了分子间的相互作用,因此模拟结果与理论结果符合良好;给出了具体的苯分子在β分子筛中扩散的相应相互作用参数值,初步揭示了大孔道分子筛中被吸附分子间相互作用的特征。     

Molecular hydrogen isotope analysis of living and fossil plants—Metasequoia as an example

Molecular hydrogen isotope analysis preformed on modern and fossil plants has made a significant impact on diverse research fields in biology and geology. Using living and fossil Metasequoia as an example, we review the technology of online GC-IRMS that made the molecular analysis of hydrogen isotope possible and discuss critical issues concerning with the studies of molecular dD and its applications. The apparent hydrogen fractionation factors between lipid molecules and source water (ewater–lipid) vary across plant taxonomy and differ among biomolecules and are affected by multiple environmental factors in which precipitation dD values exercise the first order of control. Eco-physiological factors and environmental parameters are also known to influence dD in plants. Molecular hydrogen isotope analysis of chemically stable lipid molecules, such as n-alkanes, finds a wide range of applications in detecting source sediments, reconstruction of paleoclimatic parameters, inference of air-mass trajectory, as well as in petroleum industry and environmental studies.     

An ordered mesoporous organosilica hybrid material with a crystal-like wall structure

Surfactant-mediated synthesis strategies are widely used to fabricate ordered mesoporous solids in the form of metal oxides, metals, carbon and hybrid organosilicas. These materials have amorphous pore walls, which could limit their practical utility. In the case of mesoporous metal oxides, efforts to crystallize the framework structure by thermal and hydrothermal treatments have resulted in crystallization of only a fraction of the pore walls. Here we report the surfactant-mediated synthesis of an ordered benzene-silica hybrid material; this material has an hexagonal array of mesopores with a lattice constant of 52.5 A, and crystal-like pore walls that exhibit structural periodicity with a spacing of 7.6 A along the channel direction. The periodic pore surface structure results from alternating hydrophilic and hydrophobic layers, composed of silica and benzene, respectively. We believe that this material is formed as a result of structure-directing interactions between the benzene-silica precursor molecules, and between the precursor molecules and the surfactants. We expect that other organosilicas and organo-metal oxides can be produced in a similar fashion, to yield a range of hierarchically ordered mesoporous solids with molecular-scale pore surface periodicity.     

Helical self-assembled polymers from cooperative stacking of hydrogen-bonded pairs

The double helix of DNA epitomizes this molecule's ability to self-assemble in aqueous solutions into a complex chiral structure using hydrogen bonding and hydrophobic interactions. Non-covalently interacting molecules in organic solvents are used to design systems that similarly form controlled architectures. Peripheral chiral centres in assemblies and chiral side chains attached to a polymer backbone, have been shown to induce chirality at the supramolecular level, and highly ordered structures stable in water are also known. However, it remains difficult to rationally exploit non-covalent interactions for the formation of chiral assemblies that are stable in water, where solvent molecules can compete effectively for hydrogen bonds. Here we describe a general strategy for the design of functionalized monomer units and their association in either water or alkanes into non-covalently linked polymeric structures with controlled helicity and chain length. The monomers consist of bifunctionalized ureidotriazine units connected by a spacer and carrying solubilizing chains at the periphery. This design allows for dimerization through self-complementary quadruple hydrogen bonding between the units and solvophobically induced stacking of the dimers into columnar polymeric architectures, whose structure and helicity can be adjusted by tuning the nature of the solubilizing side chains.     

Cingulin, a new peripheral component of tight junctions

The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.     

Water conduction through the hydrophobic channel of a carbon nanotube.

Confinement of matter on the nanometre scale can induce phase transitions not seen in bulk systems. In the case of water, so-called drying transitions occur on this scale as a result of strong hydrogen-bonding between water molecules, which can cause the liquid to recede from nonpolar surfaces to form a vapour layer separating the bulk phase from the surface. Here we report molecular dynamics simulations showing spontaneous and continuous filling of a nonpolar carbon nanotube with a one-dimensionally ordered chain of water molecules. Although the molecules forming the chain are in chemical and thermal equilibrium with the surrounding bath, we observe pulse-like transmission of water through the nanotube. These transmission bursts result from the tight hydrogen-bonding network inside the tube, which ensures that density fluctuations in the surrounding bath lead to concerted and rapid motion along the tube axis. We also find that a minute reduction in the attraction between the tube wall and water dramatically affects pore hydration, leading to sharp, two-state transitions between empty and filled states on a nanosecond timescale. These observations suggest that carbon nanotubes, with their rigid nonpolar structures, might be exploited as unique molecular channels for water and protons, with the channel occupancy and conductivity tunable by changes in the local channel polarity and solvent conditions.     

Molecular imprinting of bulk, microporous silica

Molecular imprinting aims to create solid materials containing chemical functionalities that are spatially organized by covalent or non-covalent interactions with imprint (or template) molecules during the synthesis process. Subsequent removal of the imprint molecules leaves behind designed sites for the recognition of small molecules, making the material ideally suited for applications such as separations, chemical sensing and catalysis. Until now, the molecular imprinting of bulk polymers and polymer and silica surfaces has been reported, but the extension of these methods to a wider range of materials remains problematic. For example, the formation of substrate-specific cavities within bulk silica, while conceptually straightforward, has been difficult to accomplish experimentally. Here we describe the imprinting of bulk amorphous silicas with single aromatic rings carrying up to three 3-aminopropyltriethoxysilane side groups; this generates and occupies microporosity and attaches functional organic groups to the pore walls in a controlled fashion. The triethoxysilane part of the molecules' side groups is incorporated into the silica framework during sol-gel synthesis, and subsequent removal of the aromatic core creates a cavity with spatially organized aminopropyl groups covalently anchored to the pore walls. We find that the imprinted silicas act as shape-selective base catalysts. Our strategy can be extended to imprint other functional groups, which should give access to a wide range of functionalized materials.     

Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption

Aquaporin-1 (AQP1) is a water channel protein expressed widely in vascular endothelia, where it increases cell membrane water permeability. The role of AQP1 in endothelial cell function is unknown. Here we show remarkably impaired tumour growth in AQP1-null mice after subcutaneous or intracranial tumour cell implantation, with reduced tumour vascularity and extensive necrosis. A new mechanism for the impaired angiogenesis was established from cell culture studies. Although adhesion and proliferation were similar in primary cultures of aortic endothelia from wild-type and from AQP1-null mice, cell migration was greatly impaired in AQP1-deficient cells, with abnormal vessel formation in vitro. Stable transfection of non-endothelial cells with AQP1 or with a structurally different water-selective transporter (AQP4) accelerated cell migration and wound healing in vitro. Motile AQP1-expressing cells had prominent membrane ruffles at the leading edge with polarization of AQP1 protein to lamellipodia, where rapid water fluxes occur. Our findings support a fundamental role of water channels in cell migration, which is central to diverse biological phenomena including angiogenesis, wound healing, tumour spread and organ regeneration.     

Electrostatic orientation during electron transfer between flavodoxin and cytochrome c

Various studies have shown that reaction rates between reversibly binding electron transfer proteins depend strongly on solution ionic strength. These observations suggest that intermolecular electrostatic interactions are important in facilitating the formation of a productive reaction complex. A recently examined system involves the reduction of vertebrate cytochrome c by bacterial flavodoxin. Although this is a nonphysiological reaction, it proceeds with rates typical for natural partners and is similarly inhibited at high ionic strengths. Here we describe computational studies which examine the role of electrostatics in the formation of a putative reaction complex between flavodoxin and cytochrome c. The results suggest that electrostatic interactions preorient the molecules before they make physical contact, facilitating the formation of an optimal reaction complex.     

煤层气分子成泡的微观过程研究

运用化学理论对煤层气分子(主要成分为甲烷)在地层水的溶解和成泡过程进行了描述,对成泡过程的受力与做功进行分析,并结合生产实际讨论了温度、生产压差、地层水矿化度、压力降和孔隙介质的大小对甲烷分子成泡规律的影响.研究表明:甲烷分子在水中成泡包括甲烷分子在水中的溶解、局部聚集成核、成泡与气泡的破裂和气泡的生长与稳定4个过程.上覆压力越小,开发的压力降越大越有利于甲烷分子脱附后形成稳定的气泡;在煤层气开发初期,甲烷以扩散方式运移,低温和低矿化度有利于气泡的生成.     

Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus

Fusion of biological membranes is mediated by specific lipid-interacting proteins that induce the formation and expansion of an initial fusion pore. Here we report the crystal structure of the ectodomain of the Semliki Forest virus fusion glycoprotein E1 in its low-pH-induced trimeric form. E1 adopts a folded-back conformation that, in the final post-fusion form of the full-length protein, would bring the fusion peptide loop and the transmembrane anchor to the same end of a stable protein rod. The observed conformation of the fusion peptide loop is compatible with interactions only with the outer leaflet of the lipid bilayer. Crystal contacts between fusion peptide loops of adjacent E1 trimers, together with electron microscopy observations, suggest that in an early step of membrane fusion, an intermediate assembly of five trimers creates two opposing nipple-like deformations in the viral and target membranes, leading to formation of the fusion pore.     

初始含水率对重塑黏土孔压特性影响试验研究

针对具有不同初始含水率的2种重塑黏土进行三轴固结不排水剪切试验(CU试验),得到了不同初始含水率重塑土三轴不排水剪切试验过程中孔隙水压力与轴向应变的关系曲线.通过对孔隙水压力分别随平均正应力和偏应力的变化规律进行深入研究,发现不同初始含水率重塑土归一化孔压u/p’0与有效应力比η之间均呈很好的线性关系.同时采用Balasubramaniam提出的归一化方法能够很好地将给定初始含水率不同固结压力下的孔压曲线进行归一化;而初始含水率与u/p0-η曲线的斜率C之间则呈近似线性减小的关系.最终基于Wood提出的孔压公式,提出了考虑初始含水率影响的孔压系数a的确定方法.