Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Common Themes in Glycoconjugate Assembly Using the Biogenesis of O-Antigen Lipopolysaccharide as a Model System

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipid-linked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this mini-review.

On thin ice: surface order and disorder during pre-melting

The effect of temperature on the structure of the ice Ih (0001) surface is considered through a series of molecular dynamics simulations on an ice slab. At relatively low temperatures (200K) a small fraction of surface self-interstitials (i.e. admolecules) appear that are formed exclusively from molecules leaving the outermost bilayer. At higher temperatures (ca. 250 K), vacancies start to appear in the inner part of the outermost bilayer exposing the underlying bilayer and providing sites with a high concentration of the dangling hydrogen bonds. Around 250-260 K aggregates of molecules formed on top of the outermost bilayer from self-interstitials become more mobile and have diffusivities approaching that of liquid water. At similar to 270-280 K the inner bilayer of one surface noticeably destructures and it appears that at above 285 K both surfaces are melting. The observed disparity in the onset of melting between the two sides of the slab is rationalised by considering the relationship between surface energy and the spatial distribution of protons at the surface; thermodynamic stability is conferred on the surface by maximising separations between dangling protons at the crystal exterior. Local hotspots associated with a high dangling proton density are suggested to be susceptible to pre-melting and may be more efficient at trapping species at the external surface than regions with low concentrations of protons thus potentially helping ice particles to catalyse reactions. A preliminary conclusion of this work is that only about 10-20 K below the melting temperature of the particular water potential employed is major disruption of the crystalline lattice noted which could be interpreted as being "liquid", the thickness of this film being about a nanometre.

Electrocatalytic activity of Ru-modified Pt(111) electrodes toward CO oxidation

The electrochemical deposition of Ru on Pt(111) electrodes has been investigated by electron diffraction, Auger spectroscopy, and cyclic voltammetry in a closed UHV transfer system. At small coverages Ru formed a monatomic commensurate layer, at higher coverage mostly small islands with a bilayer height were detected. When the Pt was almost completely covered by Ru, three-dimensional clusters developed. The island structure of Ru changed upon electrooxidation of CO, reflecting an enhanced mobility of Ru. Adsorption and electrooxidation of CO have been studied on such Ru-modified Pt(111) electrodes using cyclic voltammetry and in situ FTIR spectroscopy. Compared to the pure metals, the Ru-CO bond is weakened, the Pt-CO bond strengthened on the modified electrodes. The catalytic activity of the Ru/Pt(111) electrode toward CO adlayer oxidation is higher than that of pure Ru and a PtRu alloy (50:50). It is concluded that the electrooxidation of CO takes place preferentially at the Ru islands, while CO adsorbed on Pt migrates to them. © 1999 American Chemical Society.

A simple and low-cost method for the preparation of self-supported TiO2-WO3 ceramic heterojunction wafers

Robust, bilayer heterojunction photodiodes of TiO₂-WO₃ were prepared successfully by a simple, low-cost powder pressing technique followed by heat-treatment. Exclusive photoirradiation of the TiO₂ side of the photodiode resulted in a rapid colour change (dark blue) on the WO₃ surface as a result of reduction of W⁶⁺ to W⁵⁺ (confirmed by X-ray photoelectron spectroscopy). This colour was long lived and shown to be stable in a dry environment in air for several hours. A similar photoirradiation experiment in the presence of a mask showed that charge transfer across the heterojunction occurred approximately normal to the TiO₂ surface, with little smearing out of the mask image. As a result of the highly efficient vectorial charge separation, the photodiodes showed a tremendous increase in photocatalytic activity for the degradation of stearic acid, compared to wafers of the respective individual materials when tested separately.

Metallosupramolecular Materials for Electronic Applications: Molecular Boolean Computation

It is an exciting era for molecular computation because molecular logic gates are being pushed in new directions. The use of sulfur rather than the commonplace nitrogen as the key receptor atom in metal ion sensors is one of these directions; plant cells coming within the jurisdiction of fluorescent molecular thermometers is another, combining photochromism with voltammetry for molecular electronics is yet another. Two-input logic gates benefit from old ideas such as rectifying bilayer electrodes, cyclodextrin-enhanced room-temperature phosphorescence, steric hindrance, the polymerase chain reaction, charge transfer absorption of donor–acceptor complexes and lectin–glycocluster interactions. Furthermore, the concept of photo-uncaging enables rational ways of concatenating logic gates. Computational concepts are also applied to potential cancer theranostics and to the selective monitoring of neurotransmitters in situ. Higher numbers of inputs are also accommodated with the concept of functional integration of gates, where complex input–output patterns are sought out and analysed. Molecular emulation of computational components such as demultiplexers and parity generators/checkers are achieved in related ways. Complexity of another order is tackled with molecular edge detection routines.

Curvature of clathrin-coated pits driven by epsin

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

Interference cancellation for distributed wireless systems

O tema principal desta tese é o problema de cancelamento de interferência para sistemas multi-utilizador, com antenas distribuídas. Como tal, ao iniciar, uma visão geral das principais propriedades de um sistema de antenas distribuídas é apresentada. Esta descrição inclui o estudo analítico do impacto da ligação, dos utilizadores do sistema, a mais antenas distribuídas. Durante essa análise é demonstrado que a propriedade mais importante do sistema para obtenção do ganho máximo, através da ligação de mais antenas de transmissão, é a simetria espacial e que os utilizadores nas fronteiras das células são os mais bene ciados. Tais resultados são comprovados através de simulação. O problema de cancelamento de interferência multi-utilizador é considerado tanto para o caso unidimensional (i.e. sem codi cação) como para o multidimensional (i.e. com codi cação). Para o caso unidimensional um algoritmo de pré-codi cação não-linear é proposto e avaliado, tendo como objectivo a minimização da taxa de erro de bit. Tanto o caso de portadora única como o de multipla-portadora são abordados, bem como o cenário de antenas colocadas e distribuidas. É demonstrado que o esquema proposto pode ser visto como uma extensão do bem conhecido esquema de zeros forçados, cuja desempenho é provado ser um limite inferior para o esquema generalizado. O algoritmo é avaliado, para diferentes cenários, através de simulação, a qual indica desempenho perto do óptimo, com baixa complexidade. Para o caso multi-dimensional um esquema para efectuar "dirty paper coding" binário, tendo como base códigos de dupla camada é proposto. No desenvolvimento deste esquema, a compressão com perdas de informação, é considerada como um subproblema. Resultados de simulação indicam transmissão dedigna proxima do limite de Shannon.

On the solvation in lipid bilayers measured by pyrene fluorescence: thermal and molecular composition influence on equivalent polarity in lipidic mixtures

Phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol (CHOL) are major constituents of mammalian cell membranes. DPPC/CHOL and DPPC/DMPC are well-known binary mixtures. POPC/CHOL, DOPC/CHOL, egg-SM/CHOL, egg-SM/POPC and egg-SM/DOPC are less studied, but also important for the comprehension of the POPC/egg-SM/CHOL mixtures. These provide complex media for which polarity is hard to access. It is mainly determined by the water penetrating the bilayer (unevenly distributed creating a polarity gradient), though the influence of the dipoles from phospholipids (e.g. –PO, –CO, –OH) and the double bond in the steroid ring of CHOL cannot be neglected. CHOL derivatives are an interesting tool to verify the influence of the double bonds in the polarization of its surroundings. Pyrene fluorescence was used to access an equivalent polarity (associated to the dielectric constant) near the lipid/water interface of lipid bilayers. POPC/CHOL and DOPC/CHOL have similar thermal behavior and variation with CHOL content, though for lower CHOL content the equivalent polarity is higher for the DOPC/CHOL mixtures. The studies with DPPC and DMPC showed that pyrene does not seem to have a marked preference for either ordered or disordered phases. For DPPC/CHOL and egg-SM/CHOL the highlight goes to the behavior of the mixtures at higher CHOL amounts, where there is a substantial change in the thermal behavior and polarity values especially for the egg-SM/CHOL mixture. Egg-SM/POPC and egg-SM/DOPC show different behavior depending on which phospholipid has a higher molar proportion. The ternary mixtures analyzed do not exhibit significant differences, though there is the indication of the existence of a more ordered environment at lower temperatures and a less ordered environment for higher temperatures. The presence of 7DHC or DCHOL in egg-SM bilayers showed a tendency for the same behavior detected upon mixing higher amounts of CHOL.

Self-mated tribological systems based on multilayer micro/nanocrystalline CVD diamond coatings

The tribological response of multilayer micro/nanocrystalline diamond coatings grown by the hot filament CVD technique is investigated. These multigrade systems were tailored to comprise a starting microcrystalline diamond (MCD) layer with high adhesion to a silicon nitride (Si3N4) ceramic substrate, and a top nanocrystalline diamond (NCD) layer with reduced surface roughness. Tribological tests were carried out with a reciprocating sliding configuration without lubrication. Such composite coatings exhibit a superior critical load before delamination (130–200 N), when compared to the mono- (60–100 N) and bilayer coatings (110 N), considering ∼10 µm thick films. Regarding the friction behaviour, a short-lived initial high friction coefficient was followed by low friction regimes (friction coefficients between 0.02 and 0.09) as a result of the polished surfaces tailored by the tribological solicitation. Very mild to mild wear regimes (wear coefficient values between 4.1×10−8 and 7.7×10−7 mm3 N−1 m−1) governed the wear performance of the self-mated multilayer coatings when subjected to high-load short-term tests (60–200 N; 2 h; 86 m) and medium-load endurance tests (60 N; 16 h; 691 m).

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

The structural effects of divalent cations and insulin on phospholipid model membranes /

It is well accepted that structural studies with model membranes are of considerable value in understanding the structure of biological membranes. Many studies with models of pure phospholipids have been done; but the effects of divalent cations and protein on these models would make these studies more applicable to intact membrane. The present study, performed with above view, is a structural analysis of divalent io~cardio1ipin complexes using the technique of x-ray diffraction. Cardiolipin, precipitated from dilute solution by divalent ionscalcium, magnesium and barium, contains little water and the structure formed is similar to the structure of pure cardiolipin with low water content. The calcium-cardiolipin complex forms a pure hexagonal type II phase that exists from 40 to 400 C. The molar ratio of calcium and cardiolipin in the complex is 1 : 1. Cardiolipin, precipitated with magnesium and barium forms two co-existing phases, lamellar and hexagonal, the relative quantity of the two phases being dependent on temperature. The hexagonal phase type II consisting of water filled channels formed by adding calcium to cardiolipin may have a remarkable permeability property in intact membrane. Pure cardiolipin and insulin at pH 3.0 and 4.0 precipitate but form no organised structure. Lecithin/cardiolipin and insulin precipitated at pH 3.0 give a pure lamellar phase. As the lecithin/cardiolipin molar ratio changes from 93/7 to SO/50, (a) the repeat distance of the lamellar changes from 72.8 X to 68.2 A; (b) the amount of protein bound increases in such a way that cardiolipin/insulin molar ratio in the complex reaches a maximum constant value at lecithin/cardiolipin molar ratio 70/30. A structural model based on these data shows that the molecular arrangement of lipid and protein is a lipid bilayer coated with protein molecules. The lipid-protein interaction is chiefly electrostatic and little, if any, hydrophobic bonding occurs in this particular system. So, the proposed model is essentially the same as Davson-Daniellifs model of biological membrane.

Effect of the position of the double-bond in monounsaturated phospholipids on the dynamics of model membranes

Therllloelynalllics of lllodel 11lel1ll)rane systeills containing 1110nollnsaturatecl I)lloSI)holil) ids is strongly infllienced l)y the I)osition of the C==C dOlll)le })ond in tIle acyl chain. The telllI)eratllres of both chain-nlelting (TM) and La -+ HI! (TH) I)hase traIlsitions are lowered by IIp to 20°C when C==C is Inoved froln positions 6 or 11 to I)osition 9 in an 18-carl)on chain. This work is an attellll)t to ellicidate the uIlderlying Illoleclilar Illechanisllls reSI)Onsi])le for tllese draillatic tllerillodynaillic changes. Mixtllres of di-18: 1 l)hoSI)hatidylethanolanline with C==C at l)ositioIlS 6, 9, 11 were llsed, witll a sI1lall aI1lOlint of I)erdellterated tetradecanol, known to })e a gooel rel)Orter of the cllain Illoleclilar order. SI)ectral second 11I0I1lents were llsed to Illonitor tIle La -+ HII I)hase transition, which was fOllnd to ])e ])road (2-6°C), with a slight llysteresis on heatiIlg/cooling. The orientational order I)rofiles were nleasllred 1lSiIlg 2H Illiclear Illagnetic resonance and changes in these order I)rofiles between La aIld HII I)hases silow l)oth a local increase in order in the vicinity of the C==C bonds and an o\Terall decrease ill the average orientational order of the chain as a whole. These Sll])tle changes recluire })oth high-fidelity SI)ectrosCol)y and a careflll data analysis that takes into aCCOllnt the effects due to l)artiall1lagnetically-indllced orientational ordering of the l)ilayers. In tIle COIltext of SOllle recently rel)Orted cross-relaxation 11leaSlirenlents in Silllilar l)llOSI)llolil)iels, 0111' reslilts sllggest that large-anll)litllde conforlllational changes in the interior of tIle I110del 111eI11])ranes I)lay a 1110re significant role than I)reviollsly thOllght.

Measurement of forces between and within bilayers of neutral phospholipids

Phospholipids in water form lamellar phases made up of alternating layers of water and bimolecular lipid leaflets. Three complementary methods, osmotic, mechanical, and vapour pressures, were used to measure the work of removing water from lamellar phases composed of frozen dipalmitoylphosphatidylcholine ( DPPC ), melted DPPC, egg phosphatidylethanolamine or equimolar mixtures of DPPC and cholesterol ( DPPC/CHOL ), Concurrently the structural changes that resulted from this water removal were measured using X-ray diffraction. The work was divided into that which forces the bilayers together ( F ) and that which compresses the molecules together within the bilayers ( F )# A large repulsive force exists between bilayers composed of each of the lipids studied and this force increases exponentially as bilayer separation is decreased. F is affected by the nature of the head groups, conformation of the acyl chains and heterogeneity of these chains. In general all of the melted phosphatidylcholines ( melted DPPC, egg lecithin and DPPC/CHOL ) have large equilibrium separations in excess water resulting from large repulsive hydration forces between these bilayers. By comparison, egg PE has an increased attractive force, and frozen DPPC has a decreased hydration force; each results in smaller separations in water for these two lipids. The chemical potentials of the water between the bilayers for all these lipids lie on a continuum, indicating that interbilayer water cannot be characterized by two discrete states, usually referred to as "bound" or "non**bound". For all lipids studied a maximum of 25 % of the total work done on the system goes into deforming the bilayers. The method used here viii to separate repulsion from deformation, developed for us by v. A. Parsegian, provides a unique method for the measurement of lateral pressure of a bilayer and its modulus of deformability ( Y ). Lateral pressure is affected by the nature of the head group, conformation and heterogeneity of the acyl chains. For small changes in molecular surface area ( A ) near equilibrium, both melted and frozen DPPC have similar values for the deformability modulus. Thus in this regime it requires about the same force to change the angle of tilt of frozen chains as it does to compress the fluid bilayer. The introduction of cholesterol into bilayers of DPPC reduces dramatically the lateral pressure of the bilayers over a large range of molecular surface areas ( A ). The variation in the magnitude of bilayer repulsion with different phospholipids provides a basis for the mechanism of lipid segregation in mixed lipid systems and suggests that interacting heterogeneous membranes may influence or modulate the composition of the opposing membrane. The measurements of deformabilities of bilayers provides a direct comparison of them with the properties of monolayers.

Measurement of repulsive forces between charged phospholipid bilayers

Electrostatic forces between membranes containing charged lipids were assumed to play an important role in influencing interactions between membranes long before quantitative measurements of such forces were available. ~ur measurements were designed to measure electrostatic forces between layers of lecithin charged with lipi~s carrying ionizable head groups. These experiments have shown that the interactions between charged lipid bila.yere are dominated by electrostatic forces only at separations greater than 30 A. At smaller separations the repulsion between charged bilayers is dominated by strong hydration forces. The net repulsive force between egg lecithin bilayers containing various amounts of cherged lipids (phosphatidylglycerol (PG) 5,10 ano 50 mole%, phosphatidyli. nosi tol (PI) 10 mole% and sodium oleate (Na-Ol) 3,5 and 10 mole%, where mole% gives the ratio of the number of moles' of .charged lipid to the total number of moles of all lipids present in the sample) was stuoied with the help ('If the osmotic streas technique described by LeNeveu et aI, (1977). Also, the forces between pure PG were j_nvestigated in the same manner. The results have been plotted showing variation of force as a function of bilay- _ er separation dw• All curVes 90 obtained called force curves, were found to be similar in sha.pe, showing two distinct regions, one when dw<.30 A is a region cf very rapid iiivariation of force with separation ( it is the region dominated by hydre,tion force) and second when dw> 40 A is a region of very slow variation of force with separB.tion ( it is the region dominated by the electrostatic force). Between these two regions there exists a transition area in which, in most systems studied, a phase separation of lipids into fractions containing different amounts of charged groups, was observed. A qualitative analysis showed that our results were v/ell described by the simple electrostatic double -le.yer theory. For quantitative agreement between measured and calculated force curves however, the charge density for the calculations had to be taken as half of that given by the number density of charged lipids present in the lecithin bilayers. It is not clear at the moment what causes such low apparent degree of ionization among the charged head groups, and further study is needed in this area.