Thermotropic and lyotropic properties of long chain alkyl glycopyranosides: part III: pH-sensitive headgroups

As part of a series of papers, the influence of carbohydrate headgroups and aliphatic chains on the mesogenic properties of glycolipids was investigated. Alkyl glycosides with different types of aliphatic chains were synthesised. Neutral glycolipids were oxidized to their uronic acid derivatives, using the well established TEMPO-oxidation. For comparison a 6-deoxy-6-amino alkylglucopyranoside was synthesised. In addition, the thermotropic and lyotropic phase behaviour of the synthesised compounds were investigated. The thermotropism was characterised by polarising microscopy, the lyotropism by the contact preparation method.     

Synthesis and characterization of hydroxyethyl chitosan grafted by carboxyl ending DOVOB dendrimer: A novel liquid crystalline polymer

A novel chitosan derivative, hydroxyethyl chitosan grafted 3,4,5-tris[p-(n-dodecyloxy)-m-methoxybenzyloxy]benzoate (HECS-g-DOVOB) was prepared via grafting dendrimer 3,4,5-tris[p-(n-dodecyloxy)-m-methoxybenzyloxy]benzoic acid (DOVOB acid) onto hydroxyethyl chitosan (HECS). The structure of HECS-g-DOVOB was investigated by means of FTIR, ¹H NMR, UV and circular dichroism (CD). The degree of substitution was measured to be about 0.39 per glucose unit. The DOVOB acid was able to self-assemble into a thermotropic hexagonal columnar liquid crystalline phase showing fan-like texture, the aqueous HECS solution formed lyotropic cholesteric liquid crystal showing fingerprint texture. Whereas, HECS-g-DOVOB formed lyotropic cholesteric liquid crystal in concentrated DMSO solution with a planar texture, which was different to that of both DOVOB and HECS. The cholesteric pitch of HECS-g-DOVOB is much smaller than that of HECS. The fluorescence spectrum of HECS-g-DOVOB shows excimer fluorescence which is very similar to the dendrimer is a good indication for light-emitting polymeric material.     

Electronic and vibrational analysis of porphyrazine liquid-crystalline structure: Toward photochemical phase switching

The electronic and vibrational Raman spectra of octa-substituted (R = -SC₁₀H₂₁) Co- and Cu-porphyrazines are reported in their solid-state, mesophase, and isotropic liquid forms, as well as in THF solution. Their electronic spectra are composed of traditional Soret (CuS10 = 355 nm, CoS10 = 347 nm) and lower energy Q-bands (CuS10 = 669 nm, CoS10 = 639 nm), as well as a weaker, functionality-specific sulfur n → porphyrin π^∗ feature (CuS10 = 500 nm; CoS10 = 447 nm). In contrast to the broad Q-band for CoS10 in all three neat phases, the lower energy analogue for CuS10 is markedly sharper in the microcrystalline state, but similarly broadens in the mesophase, indicative of long range macrocycle π-π interactions that persist even into the liquid state. The resonance (λ = 647 nm) and off-resonance (λ = 785 nm) Raman spectra of these materials in each phase exhibit four diagnostic vibrations; the C_α-N_m stretch (∼1540-1553) cm⁻¹, C_β-C_β stretch (∼1450 cm⁻¹), C_α-C_β-N_p stretch (∼1300-1315 cm⁻¹), and C_α-C_β stretch (∼1070 cm⁻¹). For CoS10, these vibrations systematically shift to lower energy upon melting, while those for CuS10 collapse to degenerate sets. The differences in the electronic and vibrational profiles as a function of temperature suggest that the mesophase structure is governed by strong axial Co-S interactions for CoS10 which template macrocycle π-π stacking, while for CuS10 the same contacts exist, but they are phase dependent and markedly weaker. These inter-porphyrazine interactions are, therefore, responsible for the distinct differences in the melting and clearing temperatures of their respective mesophases. Finally, based on these diagnostic spectroscopic signatures, a photo-thermal, phase-switching mechanism is demonstrated with λ = 785 nm excitation at reduced temperatures, leading to the ability to spectrally monitor and phase change with a single photon source.     

Phase diagrams of monoacylated amide-linked disaccharide glycolipids

A series of monoacylated glycolipids with even-numbered acyl chain lengths ranging from saturated C11 to C15 and an unsaturated C17:1 fatty acid connected by an amide in linkage to the disaccharide head groups maltose, melibiose and lactose were synthesized. The structural polymorphism of the glycolipids was investigated using Fourier-transform infrared spectroscopy and differential scanning calorimetry for the detection of the gel to liquid-crystalline acyl chain melting behaviour and small-angle X-ray scattering for the elucidation of the physical structure of the lipid aggregates. Also, the phase morphology was studied by polarizing microscopy in contact preparations. The data clearly show the existence of uni- and multilamellar structures. Although only one acyl chain is present, there is no evidence for the existence of micelles - of spherical or of cylindrical (H_I) type - or of interdigitated phases. The preference for lamellar phases seems to be correlated with the intrinsic high conformational order of the amide linkage of these compounds which inhibits the formation of highly curved structures.     

脂肪酸多相脂质体与癌细胞膜相互作用的ESR谱研究

用电子自旋共振(ESR)技术对液晶态多烯脂肪酸多相脂质体与癌细胞膜相互作用进行了研究. 并探讨了其在抑制和杀伤癌细胞过程中可能具有的生物学意义.实验发现：油酸多相脂质体的影响使自旋标记物在Ec腹水肝癌细胞膜上的强固定化作用减弱, 弱固定化作用增强,使自旋标记物运动自由度增加.亚油酸多相脂质体的影响使自旋标记物在乳腺癌细胞膜上的强固定化作用增强, 弱固定化作用减弱,使自旋标记物运动自由度受到限制.蓖麻酸多相脂质体的影响使自旋标记物在S₁₈₀实体瘤细胞膜上的强固定化作用增强, 弱固定化作用减弱,使自旋标记物运动自由度受到限制.结果表明,多烯脂肪酸多相脂质体作用于膜蛋白引起了膜蛋白构象的变化.     

Conformation and packing properties of phosphatidic acid: The crystal structure of monosodium dimyristoylphosphatidate

The conformation and molecular packing of monosodium 1,2-dimyristoyl-sn-glycerophosphate (DMPA) has been determined by single crystal analysis (R = 0.107). The lipid crystallizes in the space group P2₁ with unit cell dimensions: $\text{a = 5.44, b = 7.95, c = 43.98}\text{A}\text{?}$ and β = 114.2°. The two molecules of the unit cell are related by a two-fold screw axis and pack tail-to-tail in a bilayer structure. The monosodium phosphate group packs with rather a small cross-section (24 Ų) relative to the two hydrocarbon chains. This unbalance in packing cross-section is overcome by an interdigitation of the phosphate head groups of adjacent bilayers and the formation of a single, common phosphate group layer at the bilayer interfaces. The phosphate groups are linked by hydrogen bonds to linear strands which laterally are separated by strands of sodium ions. The conformation of the molecules differs from that of other phospholipids. The glycerol chain is oriented parallel (instead of perpendicular) to the layer surface and the parallel stacking of the hydrocarbon chains is achieved by a bend of the γ-chain (instead of the β-chain). Otherwise the conformation of the glycerol dicarboxyl ester group displays the same preferred features as generally found in glycerophospholipids. The hydrocarbon chains pack according to the triclinic (T_∥) packing mode. The interaction and packing principles of the phosphate head group are discussed in relation to the structural behaviour of phosphatidic acid.     

Structural polymorphism of liquid-crystalline dispersions of double-stranded DNA complexed with synthetic polycations

Linear double-stranded DNA molecules interact with positively charged polyconidine molecules in aqueous salt solutions to yield liquid-crystalline dispersions (LCDs) with a mean particle diameter of ～6000 Å. The packing density of (DNA-polycation) complexes differs among LCD particles formed at different ionic strengths. X-ray data on the liquid-crystalline phases of (DNA-polyconidine) complexes formed under different conditions were compared with a phase diagram, reflecting polymorphism of liquid crystals of linear double-stranded DNA. It was shown that LCD was hexagonal at 0.15 M ≤ C _NaCl < 0.4 M and cholesteric at 0.4 M ≤ C _NaCl < 0.55 M. Cholesteric LCD displayed abnormal optical activity in the circular dichroism spectrum. A similar situation was observed with poly(2,5-ionene), another polycation differing in chemical structure from polyconidine. The results demonstrated structural polymorphism of (DNA-polycation) LCDs. It was assumed that the packing mode of (DNA-polycation) complexes in LCD particles can be regulated by changing NaCl concentration. The mechanism generating the cholesteric liquid-crystalline state of DNA in a narrow range of NaCl concentrations is discussed.     

Efficient production of mannosylerythritol lipids with high hydrophilicity by <Emphasis Type="Italic">Pseudozyma hubeiensis</Emphasis> KM-59

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known because of their multifunctionality and biocompatibility. A previously isolated yeast strain, Pseudozyma sp. KM-59, mainly produced a hydrophilic MEL, namely MEL-C (4-O-[4′-O-acetyl-2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-d-erythritol). In this study, we taxonomically characterize the strain in detail and investigate the culture conditions. The genetic, morphological, and physiological characteristics of the strain coincided well with those of Pseudozyma hubeiensis. On batch culture for 4 days under optimal conditions, the yield of all MELs was 21.8 g/l; MEL-C comprised approximately 65% of the all MELs. Consequently, on fed-batch culture for 16 days, the yield reached 76.3 g/l; the volumetric productivity was approximately 4.8 g l⁻¹ day⁻¹. We further examined the surface-active and self-assembling properties of the hydrophilic MELs produced by the yeast strain. They showed higher emulsifying activities against soybean oil and a mixture of hydrocarbons (2-methylnaphtarene and hexadecane, 1:1) than the synthetic surfactants tested. On water penetration scans, they efficiently formed lyotropic liquid crystalline phases such as myelines and lamella () in a broad range of their concentrations, indicating higher hydrophilicity than conventional MELs. More interestingly, there was little difference in the liquid crystal formation between the crude product and purified MEL-C. The present glycolipids with high hydrophilicity are thus very likely to have practical potential without further purification and to expand the application of MELs especially their use in washing detergents and oil-in-water-type emulsifiers.     

微乳液液晶相中脂肪酶的催化特性研究

本文探索了一种在非水介质中对酶进行固定化的新方法.研究了包埋于“水/AOT/异辛烷”系统中与有机溶剂共存的液晶相(L+LC)中的脂肪酶催化橄榄油水解的特性,发现于最适温度28℃,最佳pH为7.2,组成为(w/w%):AOT14.0%、水55.9%、异辛烷15.1%、橄榄油15.0%的条件下,脂肪酶活力较高,并且具有相当好的稳定性,尤其是产物分离和酶的回收简单易行,具有潜在的工业应用前景.     

Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins

A simple spectral editing procedure is described that generates separate subspectra for the methyl ¹³C-¹H₃ multiplet components of ¹H-¹³C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields ¹H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of ¹H-¹³C J and dipolar couplings. The editing procedure is illustrated for a Ca²⁺-calmodulin sample in isotropic and liquid crystalline phases.     

Simple multidimensional NMR experiments to obtain different types of one-bond dipolar couplings simultaneously

In order to measure residual dipolar couplings, the molecule under study has to be partially oriented in the presence of the magnetic field. It has been observed that some protein samples are not stable under the conditions imposed by the orienting media. If different types of dipolar couplings are measured sequentially, their values will not agree with a unique alignment tensor that is changing slowly over time. This could bias the structure calculation. It would be more appropriate to obtain different types of dipolar couplings simultaneously, such that all the data correspond to one effective alignment tensor. We describe here a general NMR strategy designed to do so, that can be adapted to various existing pulse sequences.     

Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.     

Structure and assembly properties of the yeast prion Ure2p

The non-Mendelian phenotype [URE3] is due to a transmissible conformational change of the protein Ure2. The infectious protein form of Ure2p has lost its function and gained the capacity to transform the active form of the protein into an inactive form. The molecular basis of this conversion process is unknown. There are however indications that the conformational changes at the origin of the propagation of the inactive form of Ure2p in yeast cells are similar to those at the origin of the transition of PrPC into the scrapie-associated PrPSc form of the protein. To better understand the nature of the conformational changes at the origin of prion propagation, we have purified, characterized biochemically, examined the assembly properties and solved the crystal structure of Ure2p. Our data are presented below and a number of conclusions dealing with the molecular basis of the conversion of soluble Ure2p into its amyloid-forming state are derived.     

龙胆草地上部分的化学成分

为研究龙胆草（Gentiana scabra Bunge）地上部分的化学成分,利用硅胶柱色谱对其乙醇提取物进行分离纯化,根据理化性质和波谱方法鉴定了化合物的结构。分离得到β-香树脂醇（1）、β-谷甾醇（2）、β-香树脂醇乙酸酯（3）、乌苏醇（4）、齐墩果酸（5）、6-去甲氧基-7-甲基茵陈色原酮（6）。其中化合物6为首次从龙胆科植物中分离得到的具有2-苯氧基色原酮骨架的天然酚类成分,本文对其1D和2DNMR谱的特征及EI质谱主要碎片的可能裂解方式进行了讨论,利用单晶X-射线衍射方法对其晶体结构进行了分析,为该类天然产物的结构表征提供了依据。     

Anti-CRISPR蛋白AcrVA2的结构生物学研究

大多数古生菌及半数细菌都含有成簇有规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)和CRISPR相关(CRISPR-associated,Cas)蛋白质构成的适应性免疫系统,来抵御外界噬菌体的入侵.而噬菌体为了对抗这种免疫系统,也进化出许多抗CRISPR (anti-CRISPR,Acr)的蛋白质,使得CRISPR-Cas系统受到抑制.来自牛眼莫拉氏菌(Moraxella bovoculi)的AcrVA2是目前发现的可抑制V-A型CRISPR-Cas系统效应蛋白Cas12a发挥切割活性的Acr蛋白之一,其作用机理尚不清楚.本文解析了自由状态的AcrVA2和MbCas12a^620-636-AcrVA2复合物的晶体结构,发现AcrVA2蛋白采用了一种新的α-β折叠结构,且只与自由状态的Cas12a结合.此外,AcrVA2与MbCas12a^620-636的结合主要依靠氢键和盐桥的相互作用力,并通过疏水界面得到进一步稳定.这些结果提示,AcrV...     

Transition-state complex of the purine-specific nucleoside hydrolase of T. vivax: enzyme conformational changes and implications for catalysis

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax have previously been solved in complex with inhibitors or a substrate. All these structures show the dimeric T. vivax nucleoside hydrolase with an "open" active site with a highly flexible loop (loop 2) in its vicinity. Here, we present the crystal structures of the T. vivax nucleoside hydrolase with both soaked (TvNH-ImmH(soak)) and co-crystallised (TvNH-ImmH(co)) transition-state inhibitor immucillin H (ImmH or (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol) to 2.1 A and 2.2 A resolution, respectively. In the co-crystallised structure, loop 2 is ordered and folds over the active site, establishing previously unobserved enzyme-inhibitor interactions. As such this structure presents the first complete picture of a purine-specific NH active site, including leaving group interactions. In the closed active site, a water channel of highly ordered water molecules leads out from the N7 of the nucleoside toward bulk solvent, while Trp260 approaches the nucleobase in a tight parallel stacking interaction. Together with mutagenesis results, this structure rules out a mechanism of leaving group activation by general acid catalysis, as proposed for base-aspecific nucleoside hydrolases. Instead, the structure is consistent with the previously proposed mechanism of leaving group protonation in the T. vivax nucleoside hydrolase where aromatic stacking with Trp260 and an intramolecular O5'-H8C hydrogen bond increase the pKa of the N7 sufficiently to allow protonation by solvent. A mechanism that couples loop closure to the positioning of active site residues is proposed based on a comparison of the soaked structure with the co-crystallized structure. Interestingly, the dimer interface area increases by 40% upon closure of loop 2, with loop 1 of one subunit interacting with loop 2 of the other subunit, suggesting a relationship between the dimeric form of the enzyme and its catalytic activity.     

NMR investigations of interactions between anesthetics and lipid bilayers

Interactions between anesthetics (lidocaine and short chain alcohols) and lipid membranes formed by dimyristoylphosphatidylcholine (DMPC) were studied using NMR spectroscopy. The orientational order of lidocaine was investigated using deuterium NMR on a selectively labelled compound whereas segmental ordering in the lipids was probed by two-dimensional ¹H-¹³C separated local field experiments under magic-angle spinning conditions. In addition, trajectories generated in molecular dynamics (MD) computer simulations were used for interpretation of the experimental results. Separate simulations were carried out with charged and uncharged lidocaine molecules. Reasonable agreement between experimental dipolar interactions and the calculated counterparts was observed. Our results clearly show that charged lidocaine affects significantly the lipid headgroup. In particular the ordering of the lipids is increased accompanied by drastic changes in the orientation of the P-N vector in the choline group.     

On the similarity of properties in solution or in the crystalline state: A molecular dynamics study of hen lysozyme

As protein crystals generally possess a high water content, it is assumed that the behaviour of a protein in solution and in crystal environment is very similar. This assumption can be investigated by molecular dynamics (MD) simulation of proteins in the different environments. Two 2ns simulations of hen egg white lysozyme (HEWL) in crystal and solution environment are compared to one another and to experimental data derived from both X-ray and NMR experiments, such as crystallographic B-factors, NOE atom–atom distance bounds, ³J_{H N}-coupling constants, and ¹H-¹⁵N bond vector order parameters. Both MD simulations give very similar results. The crystal simulation reproduces X-ray and NMR data slightly better than the solution simulation.     

Investigating the change in the photophysical properties of a trio of tetraphenylcyclopentadienone derivatives with varied groups on the aromatic rings in the 3‐ and 4‐positions

Organic compounds with electronic properties, such as a small band gap, are useful in areas ranging from organic field effect transistors to solar cells. Such organic compounds can possess conjugation and/or aromatic systems, with one example being tetraphenylcyclopentadienone and its derivatives. A trio of dramatically coloured tetraphenylcyclopentadienone derivatives with varied substituents on the aromatic rings in the 3‐ and 4‐positions were prepared. Their identities were confirmed using the usual methods, for example ¹H nuclear magnetic resonance (NMR) spectroscopy, and their purity quantified using elemental analysis. The X‐ray crystal structure of compound 2 was determined. Its notable structural features involved the cyclopentadienone core with its distinct C?C and C=C bond lengths and its overall nonplanarity, both of which served to mitigate its antiaromatic nature. Chloroform solutions of compounds 2 – 4 exhibited absorption spectra with three absorption bands at approximately 250, 350, and 500 nm that were assigned to (π)→(π*) transitions. Computational chemistry methods assisted in assigning the observed transitions to a specific molecular orbital combination in the structures of 2 – 4 . Emission in the red end of the visible spectrum (550–625 nm) was observed from chloroform solutions of all three of the prepared compounds.     

Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala

Mitochondrial ferritin is a recently identified protein precursor encoded by an intronless gene. It is specifically taken up by the mitochondria and processed to a mature protein that assembles into functional ferritin shells. The full mature recombinant protein and its S144A mutant were produced to study structural and functional properties. They yielded high quality crystals from Mg(II) solutions which diffracted up to 1.38 Angstrom resolution. The 3D structures of the two proteins resulted very similar to that of human H-ferritin, to which they have high level of sequence identity (approximately 80%). Metal-binding sites were identified in the native crystals and in those soaked in Mn(II) and Zn(II) solutions. The ferroxidase center binds binuclear iron at the sites A and B, and the structures showed that the A site was always fully occupied by Mg(II), Mn(II) or Zn(II), while the occupancy of the B site was variable. In addition, distinct Mg(II) and Zn(II)-binding sites were found in the 3-fold axes to block the hydrophilic channels. Other metal-binding sites, never observed before in H-ferritin, were found on the cavity surface near the ferroxidase center and near the 4-fold axes. Mitochondrial ferritin showed biochemical properties remarkably similar to those of human H-ferritin, except for the difficulty in renaturing to yield ferritin shells and for a reduced ( approximately 41%) rate in ferroxidase activity. This was partially rescued by the substitution of the bulkier Ser144 with Ala, which occurs in H-ferritin. The residue is exposed on a channel that connects the ferroxidase center with the cavity. The finding that the mutation increased both catalytic activity and the occupancy of the B site demonstrated that the channel is functionally important. In conclusion, the present data define the structure of human mitochondrial ferritin and provide new data on the iron pathways within the H-type ferritin shell.