An inverse face-centered cubic phase formed by diacylglycerol-phosphatidylcholine mixtures

Fully hydrated unsaturated diacylglycerol-phosphatidylcholine mixtures are found to adopt an inverse face-centered cubic phase, of crystallographic cubic aspect 15. The same behavior is observed for either the 1,2- or 1,3-isomer of the diacylglycerol. This Q15 cubic phase, of probable space group Fd3m (Q227), occurs between an inverse hexagonal (HII) phase and an inverse micellar (L2) solution, with increasing diacylglycerol concentration, which implies that the mean curvature of the interface is more negative than that of the HII phase. This behavior is quite different from that of the more usual bicontinuous inverse cubic phases Pn3m (Q224), Im3m (Q229), and Ia3d (Q230), which normally occur between the lamellar L alpha and the HII phases. One possible structure for the Fd3m cubic phase has recently been proposed (Mariani, P., Luzzati, V., & Delacroix, H. (1988) J. Mol. Biol. 204, 165-189), consisting of tetrahedrally arranged clusters of inverse micelles surrounded by a continuous cage of tetrahedrally connected water/lipid (inverse) channels.     

Cubic phases of lipid-containing systems. The structure of phase Q223 (space group Pm3n). An X-ray scattering study.

The hexagonal (H) and the cubic (Q223) phases of the systems dodecyltrimethylammonium chloride-water and palmitoyllysophosphatidy choline-water have been studied by X-ray scattering techniques. The signs of the reflections of phase H were determined by a systematic study as a function of the water content, those of phase Q223 were assessed using a pattern recognition approach based upon the axiom that the histograms of the electron density maps of phases Q223 and H, extrapolated to the same concentration and properly normalized in scale and shape, are very similar to each other. In the case of phase Q223, all the sign combinations (the phi-sets) compatible with the observed reflections were generated, and each of the corresponding histograms was compared with the histogram of the map of phase H. One novelty of this work is the use of a highly sensitive criterion to estimate the similarity of the histograms, namely the distance in the six-dimensional space of the moments [mean value of (delta rho)n]1/n, for 3 greater than or equal to n greater than or equal to 8. In the two systems, the use of this criterion has led to the unambiguous choice of one electron density map. The maps show that the structure of phase Q223 consists of disjointed micelles (of type I), belonging to two different classes: those of one class are quasi-spherical in shape and are centered at the points a, those of the other class are disc-shaped and are centred at the points c. The results of this work rule out a structure formed by a cage-like distribution of rods enclosing a set of quasi-spherical micelles and is consistent with previous proposals. This is the second example, after that of phase Q227, of a micellar cubic phases in lipid-containing systems; all the known examples of phase Q223 are of type I, those of phase Q227 of type II.     

Interaction of the peptide antibiotic alamethicin with bilayer- and non-bilayer-forming lipids: influence of increasing alamethicin concentration on the lipids supramolecular structures

Incorporation of the helical antimicrobial peptide alamethicin from aqueous phase into hydrated phases of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) was investigated within a range of peptide concentrations and temperatures by time-resolved synchrotron X-ray diffraction. It was found that alamethicin influences the organizations of the non-bilayer-forming (DOPE) and the bilayer-forming (DOPC) lipids in different ways. In DOPC, only the bilayer thickness was affected, while in DOPE new phases were induced. At low peptide concentrations (<1.10(-4) M), an inverted hexagonal (H(II)) phase was observed as with DOPE dispersions in pure buffer solution. A coexistence of two cubic structures was found at the critical peptide concentration for induction of new lipid/peptide phases. The first one Q224 (space group Pn3m) was identified within the entire temperature region studied (from 1 to 45 degrees C) and was found in coexistence with H(II)-phase domains. The second lipid/peptide cubic structure was present only at temperatures below 16 degrees C and its X-ray reflections were better fitted by a Q212 (P4(3)32) space group, rather than by the expected Q229 (Im3m) space group. At alamethicin concentrations of 1 mM and higher, a nonlamellar phase transition from a Q224 cubic phase into an H(II) phase was observed. Within the investigated range of peptide concentrations, lamellar structures of two different bilayer periods were established with the bilayer-forming lipid DOPC. They correspond to lipid domains of associated and nonassociated helical peptide. The obtained X-ray results suggest that the amphiphilic alamethicin molecules adsorb from the aqueous phase at the lipid head group/water interface of the DOPE and DOPC membranes. At sufficiently high (>1.10(-4) M) solution concentrations, the peptide is probably accommodated in the head group region of the lipids thus inducing structural features of mixed lipid/peptide phases.     

Polymorphism of a lipid extract from Pseudomonas fluorescens: structure analysis of a hexagonal phase and of a novel cubic phase of extinction symbol Fd--

The phase diagram of the Pseudomonas fluorescens lipid extract is unusual, in the sense that it displays a cubic phase straddled by a hexagonal phase. The hexagonal phase was studied over an extended concentration range, and the reflections were phased on the assumption that the structure contains circular cylinders of known radius. The cubic phase, whose extinction symbol is Fd--, was analyzed by reference to space group No. 227 (Fd3m). The phases of the reflections were determined by using a novel pattern recognition approach, based upon the notion that the average fourth power of the electron density contrast mean value of (delta r)4 is dependent on chemical composition but not on physical structure, provided that the function delta r(r) satisfies the constraints mean value of (delta r) = 0 and mean value of (delta r)2 = 1. As a further constraint, a shape normalization is used, in the form of a Gaussian apodization of the intensities, which has the effect of normalizing the curvature of the autocorrelation function at the origin. We analyzed two cubic samples of different composition: for each of them we generated all the phase combinations compatible with the X-ray scattering data and we searched for those whose mean value of (delta r)4 best agrees with the hexagonal phase. Taking advantage of the favorable properties of the phase diagram, we carefully explored the effects of various parameters; we concluded that the chemical composition of the phases being compared must be identical, that the X-ray scattering data should not be truncated artificially, and that the apodization must be mild so that the curvature takes a value intermediate between those corresponding to the raw data of the two phases. When all these precautions were taken, mean value of (delta r)4 was found to be remarkably invariant; this conclusion is important in view of the possible usefulness of the novel technique in tackling ab initio--and at very low resolution--structural problems of more general interest. The structure of the cubic phase consists of a 3D network of rods joined tetrahedrally 4 by 4 according to a diamond lattice and of a family of quasi-spherical disjointed micelles; the core of the rods and of the micelles is polar, and the interstices are filled by the hydrocarbon chains (structure of type II). All the dimensions (diameter of rods and micelles, area per chain at the polar/apolar interface) are consistent with the chemical properties of the system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of electrostatic interactions on phase stability of cubic phases of membranes of monoolein/dioleoylphosphatidic acid mixtures.

To elucidate effects of electrostatic interactions resulting from surface charges on structures and phase stability of cubic phases of lipid membranes, membranes of 1-monoolein (MO) and dioleoylphosphatidic acid (DOPA) (DOPA/MO membrane) mixtures have been investigated by small-angle x-ray scattering method. As increasing DOPA concentration in the DOPA/MO membrane at 30 wt% lipid concentration, a phase transition from Q(224) to Q(229) phase occurred at 0.6 mol% DOPA, and at and above 25 mol% DOPA, DOPA/MO membranes were in the L(alpha) phase. As NaCl concentration in the bulk phase increased, for 10% DOPA/90% MO membrane in excess water, a Q(229) to Q(224) phase transition occurred at 60 mM NaCl, and then a Q(224) to H(II) phase transition occurred at 1.2 M NaCl. Similarly, for 30% DOPA/70% MO membrane in excess water, at low NaCl concentrations it was in the L(alpha) phase, but at and above 0.50 M NaCl it was in the Q(224) phase, and then at 0.65 M NaCl a Q(224) to H(II) phase transition occurred. These results indicate that the electrostatic interactions in the membrane interface make the Q(229) phase more stable than the Q(224) phase, and that, at larger electrostatic interactions, the L(alpha) phase is more stable than the cubic phases (Q(224) and Q(229)). We have found that the addition of tetradecane to the MO membrane induced a Q(224)-to-H(II) phase transition and also that to the 30% DOPA/70% MO membrane induced an L(alpha)-to-H(II) phase transition. By using these membranes, the effect of the electrostatic interactions resulting from the membrane surface charge (DOPA) on the spontaneous curvature of the monolayer membrane has been investigated. The increase in DOPA concentration in the DOPA/MO membrane reduced the absolute value of spontaneous curvature of the membrane. In the 30% DOPA/70% MO membrane, the absolute value of spontaneous curvature of the membrane increased with an increase in NaCl concentration. On the basis of these new results, the phase stability of DOPA/MO membranes can be reasonably explained by the spontaneous curvature of the monolayer membrane and a curvature elastic energy of the membrane.     

The effect of increasing concentrations of precipitating salts used to crystallize proteins on the structure of the lipidic Q224 cubic phase

A major obstacle to elucidating the structure of membrane proteins at high resolution is the difficulty of preparing these proteins as well as to grow well-ordered crystals. During the last few years several groups have considered the use of three-dimensional bicontinuous lipidic cubic phases as a possible crystallization matrix for such molecules. In a few cases these studies have been successfully approached by other laboratories, however, a number of questions remain, particularly in regard to the effects of solutes on the phase diagrams of lipid-water systems. In the present work we report the structural behavior of the lipidic Q224 (Pn3m), Q230 (Ia3d) and HII phases systematically studied in the presence of a range of concentrations of various salts and precipitating agents at various pH values. Some of the results reported here have been presented elsewhere Vargas et al. (2000) [Strategies in membrane protein crystallization. Chemical Prospectives in Crystallography of Molecular Biology. International School of Crystallography, NATO-ASI course, Erice (Italy)].     

Accelerated formation of cubic phases in phosphatidylethanolamine dispersions.

By means of x-ray diffraction we show that several sodium salts and the disaccharides sucrose and trehalose strongly accelerate the formation of cubic phases in phosphatidylethanolamine (PE) dispersions upon temperature cycling through the lamellar liquid crystalline-inverted hexagonal (Lalpha-HII) phase transition. Ethylene glycol does not have such an effect. The degree of acceleration increases with the solute concentration. Such an acceleration has been observed for dielaidoyl PE (DEPE), dihexadecyl PE, and dipalmitoyl PE. It was investigated in detail for DEPE dispersions. For DEPE (10 wt% of lipid) aqueous dispersions at 1 M solute concentration, 10-50 temperature cycles typically result in complete conversion of the Lalpha phase into cubic phase. Most efficient is temperature cycling executed by laser flash T-jumps. In that case the conversion completes within 10-15 cycles. However, the cubic phases produced by laser T-jumps are less ordered in comparison to the rather regular cubic structures produced by linear, uniform temperature cycling at 10 degrees C/min. Temperature cycles at scan rates of 1-3 degrees C/min also induce the rapid formation of cubic phases. All solutes used induce the formation of Im3m (Q229) cubic phase in 10 wt% DEPE dispersions. The initial Im3m phases appearing during the first temperature cycles have larger lattice parameters that relax to smaller values with continuation of the cycling after the disappearance of the Lalpha phase. A cooperative Im3m --> Pn3m transition takes place at approximately 85 degrees C and transforms the Im3m phase into a mixture of coexisting Pn3m (Q224) and Im3m phases. The Im3m/Pn3m lattice parameter ratio is 1. 28, as could be expected from a representation of the Im3m and Pn3m phases with the primitive and diamond infinite periodic minimal surfaces, respectively. At higher DEPE contents ( approximately 30 wt%), cubic phase formation is hindered after 20-30 temperature cycles. The conversion does not go through, but reaches a stage with coexisting Ia3d (Q230) and Lalpha phases. Upon heating, the Ia3d phase cooperatively transforms into a mixture of, presumably, Im3m and Pn3m phases at about the temperature of the Lalpha-HII transition. This transformation is readily reversible with the temperature. The lattice parameters of the DEPE cubic phases are temperature-insensitive in the Lalpha temperature range and decrease with the temperature in the range of the HII phase.     

Thermomyces lanuginosus lipase in the liquid-crystalline phases of aqueous phytantriol: X-ray diffraction and vibrational spectroscopic studies

The influence of Thermomyces lanuginosus lipase (TLL) on the phase behaviour of liquid-crystalline phases of aqueous phytantriol as well as conformational changes of TLL entrapped in the cubic Q230 phase have been studied by small angle X-ray diffraction (SAXD), FT-Raman, and FT-IR techniques. It was found that the lipidic Q230 phase is able to accommodate up to 10 wt.% of TLL, and the temperature of phase transition to the inverted hexagonal phase H(II) increases indicating stabilizing effect of the protein. FT-Raman analysis of Trp amino acid marker band W3 revealed that the average rotation angle around the C3-Cbeta bond of four Trp residues of TLL in the Q230 phase increases. Reasoning from available TLL crystallographic data, this result is explained by structural transition of entrapped protein to so-called "open" and more related to the enzymatically-active conformation. TLL secondary structure analysis by amide I and amide III vibrational bands showed that content of alpha-helixes does not change, while a part of beta-sheet structures transforms to less ordered elements upon incorporation of protein into the Q230 phase of aqueous phytantriol.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Towards redox active liquid crystalline phases of lipids: a monoolein/water system with entrapped derivatives of ferrocene

The phase and electrochemical behavior of the aqueous mixtures of monoolein (MO) and synthetic ferrocene (Fc) derivatives containing long alkyl chains-(Z)-octadec-9-enoylferrocene (1), (Z)-octadecen-9-ylferrocene (2), and ferrocenylmethyl (Z)-octadec-9-enoate (3)-were studied. At low hydration, the reversed micelles (L(2) phase) and cubic Q(230) phase of MO can accommodate relatively high amounts (>6 wt.%) of the Fc-derivative 2, whereas at high hydration, the pseudoternary cubic phase Q(224) is destabilized even at about 2 wt.% of this Fc. Increasing the Fc-derivative content induces L(alpha)-->L(2) and L(alpha)-->reversed bicontinuous cubic phase (Q(II))-->H(II) transitions depending upon hydration. A rough study of the MO system containing compounds 1 and 3 indicates very similar phase behavior to that of the MO/2/H(2)O system. Compound 2 apparently has no effect on the lipid monolayer thickness in the pseudoternary L(alpha), H(II) and Q(II) liquid crystalline phases of MO. Within a 3D-structure of the Q(224) phase, derivatives 1-3 exhibit electrochemical activity on the gold electrode. The one-electron redox conversion processes are electrochemically quasi-reversible and controlled by diffusion. The values of apparent diffusion coefficient (D(app)) and heterogeneous electron-transfer rate constant (k(s)) of Fcs are significantly lower in the cubic phase matrix when compared to the acetonitrile solution. By contrast, the MO H(II) phase with entrapped Fc-derivatives does not exhibit electrochemical activity on the electrode surface. It is suggested that the diffusional anisotropy and/or localized aggregation of compounds 1-3 within a 2D-structure of the H(II) phase account(s) for the latter observation.     

Cubic topology in surfactant and lipid mixtures

Ternary systems of palmitoyl-oleoyl-phosphatidylcholine (POPC) and the non-ionic surfactant C₁₂EO₂ (di-ethylene-oxide-mono-dodecyl-ether) in water have been studied with optical microscopy, NMR, DSC and X-rays from ambient temperatures to 45 °C. Below 29 °C the system is in the lamellar liquid crystalline state. Between 30 and 32 °C it transforms into a cubic Ia3d structure which converts into the cubic Pn3m phase at 39 °C. The transitions are fully reversible. An epitaxial relationship between all three phases was found, which is an elegant and convenient way to rearrange molecules from lamellar bilayers to a network of curved surfaces. The la3d (Q²³⁰) to Pn3m (Q²²⁴) transition occurs without measurable enthalpy change. This, together with the metric relation of 1.60 between the cubic lattice constants is strong evidence for a Bonnet transformation, where the structural changes occur without change in curvature. The potential significance of the cubic phases as intermediate structures for biological processes, e. g. transport across a bilayer or fusion of membranes, are discussed.     

Structural relationships between lamellar,cubic and hexagonal phases in monoglyceride-water systems. possibility of cubic structures in biological systems

The transitions lamellar → cubic → hexagonal in the aqueous system of sunflower oil monoglycerides are analysed. X-Ray diffraction data show linear relationships between the lattices of the three phases, which are discussed on the basis of structures formed by lipid bilayer units. The cubic structure is related to ‘Schwarz's primitive cubic minimal surface’ and consists of a three-dimensional continuous bilayer system separating two separate water channel systems.It is also pointed out that the three-dimensional membrane system in plant plastids, the prolamellar body, which is involved in the formation of thylakoid membranes of chloroplasts, has a structure which is closely related to or identical with that of the cubic phase of monoglyceride-water systems.     

The temperature-composition phase diagram and mesophase structure characterization of monopentadecenoin in water.

The temperature-composition phase diagram of monopentadecenoin, a monoacylglycerol with a cis monounsaturated fatty acid 15 carbon atoms long (C15:1c10) in water was constructed using x-ray diffraction. Low- and wide-angle diffraction patterns were collected from samples of fixed hydration as a function of temperature in the heating direction on x-ray-sensitive film. The temperature and hydration ranges investigated were 0-104 degrees C and 0-60% (w/w) water, respectively. The phases identified in the system include the lamellar crystalline phase, the lamellar liquid crystalline phase, the fluid isotropic phase, and two inverted cubic phases belonging to space groups la3d (Q230) and Pn3m (Q244). Particular attention has been devoted to the issues of phase equilibrium, phase boundary verification, and structure characterization. The phase diagrams of monopentadecenoin, monomyristolein (C14:1c9), and monoolein (C18:1c9) are compared, and the impact of molecular structure on mesophase stability and structure is discussed.     

Transport properties of rigid bent-rod macromolecules and of semiflexible broken rods in the rigid-body treatment. Analysis of the flexibility of myosin rod.

The translational diffusion coefficients, rotational relaxation times and intrinsic viscosities of rigid bent rods, composed by two rodlike arms joined rigidly at an angle alpha, have been evaluated for varying conformation using the latest advances in hydrodynamic theory. We have considered semiflexible rods in which the joint is an elastic hinge or swivel, with a potential V(alpha) = 1/2Q alpha 2 with constant Q. Accepting the rigid-body treatment, we calculate properties of broken rods by averaging alpha-dependent values for rigid rods. The results are finally used to interpret literature values of the properties of myosin rod. Q is regarded as an adjustable parameter, and the value fitted is such that the average bending angle of myosin rod is approximately 60 degrees.     

Lipid polymorphism: a correction. The structure of the cubic phase of extinction symbol Fd-- consists of two types of disjointed reverse micelles embedded in a three-dimensional hydrocarbon matrix.

The X-ray scattering study of a cubic phase of extinction symbol Fd--, recently performed on a lipid extract (PFL) from Pseudomonas fluorescens [Mariani et al. (1990) Biochemistry 29, 6799-6810] has been extended to several other systems, all consisting of mixtures of water-miscible (MO, PC, PE, oleate) and of water-immiscible (FA, DG) lipids, plus water. In all of these systems the cubic phase was observed in the presence of excess water. Some inconsistencies observed between PFL and the other systems, the fact that in PFL one of the reflections of the cubic phase happened to coincide with the strongest reflection of the hexagonal phase, and the finding, in one of the original cubic samples of PFL kept in the cold for more than 3 years, that the intensity of one of the reflections had decreased dramatically all indicated that a nonnegligible amount of a hexagonal impurity was in fact present in the samples of PFL originally thought to contain a pure cubic phase. The intensities were corrected for that impurity and analyzed again using a pattern recognition approach based upon the axiom that the histogram of the electron density maps is invariant with respect to physical structure, when different phases are compared whose chemical composition is the same. The hexagonal phase provided the reference phase for the comparison. The moments mean value of (delta rho)n were used to compare the histograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct determination of crystallographic phases for diffraction data from lipid bilayers. II. Refinement of phospholipid structures.

Using a systematic approach for the acceptance of crystallographic phase assignment, based on the evaluation of triplet structure invariants, electron and x-ray diffraction data from phospholipid multilamellar arrays are analyzed by direct methods. After calculation of Fourier maps with a partial set of phased structure factor magnitudes, the structure is refined in real space by flattening of the hydrocarbon region of the bilayer and an optimal solution is sought either by the calculation of [delta rho 4] suggested by Luzzati, where rho is the structure density or by a test of density smoothness [magnitude of delta rho/ delta r magnitude of], where r positions are located along the normal to the lamellar surface. Reanalyses of previously determined structures sometimes lead to new conclusions (e.g., a possible similarity of the electron density profile for DL-DMPE and L-DMPE, and a clear indication of the fatty acid adduct in the mixed L-DPPC/palmitic acid bilayer). Because of presumed secondary scattering perturbations (primarily to the least intense reflections), the refinements of the electron diffraction intensities are less easily evaluated than those carried out with x-ray diffraction data.     

Temperature- and pressure-dependent phase behavior of monoacylglycerides monoolein and monoelaidin.

We used x-ray and neutron diffraction to study the temperature- and pressure-dependent structure and phase behavior of the monoacylglycerides 1-monoelaidin (ME) and 1-monoolein (MO) in excess water. The monoacylglycerides were chosen for investigation of their phase behavior because they exhibit mesomorphic phases with one-, two-, and three-dimensional periodicity, such as lamellar, an inverted hexagonal and bicontinuous cubic phases, in a rather easily accessible temperature and pressure range. We studied the structure, stability, and transformations of the different phases over a wide temperature and pressure range, explored the epitaxial relations that exist between different phases, and established a relationship between the chemical structure of the lipid molecules and their phase behavior. For both systems, a temperature-pressure phase diagram has been determined in the temperature range from 0 to 100 degrees C at pressures from ambient up to 1400 bar, and drastic differences in phase behavior are found for the two systems. In MO-water dispersions, the cubic phase Pn3m extends over a large phase field in the T,p-plane. At temperatures above 95 degrees C, the inverted hexagonal phase is found. In the lower temperature region, a crystalline lamellar phase is induced at higher pressures. The phases found in ME-water include the lamellar crystalline Lc phase, the L beta gel phase, the L alpha liquid-crystalline phase, and two cubic phases belonging to the crystallographic space groups Im3m and Pn3m. In addition, the existence of metastable phases has been exploited. Between coexisting metastable cubic structures, a metric relationship has been found that is predicted theoretically on the basis of the curvature elastic energy approximation only.     

Role of residues 230 and 236 of actin in myosin-ATPase activation by actin-tropomyosin

The Dictyostelium/Tetrahymena-chimeric actin (Q228K/T229A/A230Y) showed higher Ca(2+)-activation of myosin S1 ATPase in the presence of tropomyosin-troponin. The crystal structure of the chimeric actin is almost the same as that of wild-type except the conformation of the side chain of Leu236. Here, we introduced an additional mutation (L236A), in which the side chain of Leu236 was truncated, into the chimeric actin (Q228K/T229A/A230Y/L236A). Without regulatory proteins, the new mutant actin showed normal myosin S1 activation and normal sliding velocity. However, in the presence of tropomyosin, the new mutant actin activated myosin S1 ATPase higher than the wild-type actin and showed higher velocities in in vitro motility assay at low HMM concentrations. These results suggest that the mutations of A230Y and L236A in the actin subdomain-4 facilitate the transition of thin filaments from a "closed" state to an "open" state.     

Crystal structures of [Met5] and [(4-bromo)Phe4,Met5]enkephalins: formation of a dimeric antiparallel beta-structure

The crystal structure of [(4-bromo)Phe4,Met5]enkephalin (Tyr-Gly-Gly-(4-bromo)-Phe-Met) shows two independent molecular conformations. The molecules are arranged in parallel in a head-to-tail fashion and form an antiparallel beta-sheet structure involving intermolecular hydrogen bonds. This dimeric beta-structure is also observed in the [Met5]enkephalin crystal, in spite of their different crystal packing environments, which shows the energetic stability of this molecular conformation. The three-dimensional similarity between the dimeric beta-structure and the beta-turn form is discussed in the relation to the opioid delta and mu receptors.     

Effect of Electrostatic Interactions on Phase Stability of Cubic Phases of Biomembranes

We investigated effect of electrostatic interactions due to surfacecharges on structures and stability of cubic phases of monoolein (MO)membrane using the small-angle X-ray scattering method. Firstly, wechanged the surface charge density of the membrane by usingdioleoylphosphatidic acid (DOPA). As increasing DOPA concentration in themembrane at 30 wt % lipid concentration, a Q²²⁴ to Q²²⁹ phasetransition occurred at 0.6 mol % DOPA, and at and above 25 mol %, DOPA/MOmembranes were in the L phase. NaCl in the bulk phase reduced theeffect of DOPA. These results indicate that as the electrostaticinteractions increase, the most stable phase changes as follows: Q²²⁴ Q²²⁹ L. The increase in DOPAconcentration reduced the absolute value of spontaneous curvature of themembrane, | H₀ |. Secondly, we changed the surface charge of themembrane by adding a de novo designed peptide, which has netpositive charges and a binding site on the electrically neutral membraneinterface. The peptide-1 (WLFLLKKK) induced a Q²²⁴ to Q²²⁹phase transition in the MO membrane at low peptide concentration. As NaClconcentration increases, the MO/peptide-1 membrane changed from Q²²⁹to Q²²⁴ phase. The increase in peptide-1 concentration reduced |H₀ |. Based on these results, the stability of the cubic phases and themechanism of phase transition between cubic phase and L phase arediscussed.