Synthesis and ordered structure of amphipatic block copolymers with a saccharide and a peptide block

Amphipatic block copolymers (OβEb) with a hydrophilic saccharide block and a hydrophobic polypeptide block were synthesized. In these copolymers the saccharide block is the glyco-amino acid Oβ from ovomucoid and the peptide block (Eb) is a poly(γ-benzyl-L -glutamate) block. Copolymers OβEb exhibit, in the solid state and in Me₂SO concentrated solutions, mesomorphic lamellar structures where the polypeptide chains are in an α-helical conformation. Depending on the molecular weight of the polypeptide block, three types of lamellar structures are obtained, and they differ by the mode of organization of the polypeptide chains in their lamellae and by the T or Y conformation of the saccharide block.     

Properties of ternary phospholipid/dimethyl sulfoxide/water systems at low temperatures

X-ray diffraction, neutron diffraction and differential scanning calorimetry were used to investigate phase transitions in the ternary system phospholipid/dimethyl sulfoxide (DMSO)/water under cooling for three homologous phospholipids: dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). Below the temperature of ice formation from -40 to -113 degrees C, a new lamellar phase of DPPC and DSPC was found at and above a DMSO molar fraction of X(DMSO) = 0.05. Below X(DMSO) = 0.05 only a single dehydrated Lc-phase exists after ice formation. The new phase has an increased membrane repeat distance and coexists with a dehydrated Lc-phase. DPPC with a DMSO molar fraction of X(DMSO) = 0.07 shows a membrane repeat distance of the new phase of d = 6.61 +/- 0.03 nm. The value of d increases at the increase of X(DMSO). The new phase was not observed in the ternary system with DMPC. No correlation between the new phase and the glass transition of bound water in the intermembrane space was detected. The new phase was detected only in the systems with excess of water. The creation of the new phase demonstrates the specific DMSO interaction with hydrocarbon chains.     

Gramicidin induces the formation of non-bilayer structures in phosphatidylcholine dispersions in a fatty acid chain length dependent way

The hydrophobic peptide gramicidin is shown by ³¹P-NMR, freeze-fracture electron microscopy and small-angle X-ray diffraction, to induce a hexogonal H_II-phase lipid organization when incorporated in liquid crystalline saturated and unsaturated synthetic and natural phosphatidylcholines if the length of the fatty acids exceeds a 16 carbon atoms chain. The amount of hexagonally organized lipid increases with increasing fatty acid chain length. With phosphatidylcholines possessing shorter fatty acid chains, as well as with the longer phosphatidylcholines in the gel state, a lamellar organization results. Of the various possible models to explain the induction of the hexagonal H_II phase by gramicidin, one in which gramicidin dimers span adjacent cylinders of the hexagonal H_II phase seems most plausible. In phosphatidylcholines with intermediary chain lengths gramicidin induces intermediary structures, such as lipidic particles and possibly cubic phases. These experiments suggest that the balance between the length of the hydrophobic domain of a peptide and the membrane thickness is of critical importance for the structure of the membrane. In relation to this observation, the possible involvement of non-bilayer lipid structures in insertion and anchoring of membrane proteins is discussed.     

Effect of lysophosphatidylcholine on behavior and structure of phosphatidylcholine liposomes

Differential scanning calorimetry (DSC), fluorescence polarization and X-ray diffraction were per-formed to investigate the kinetics of the micellar to the lamellar phase transition of dipalmitoylphosphatidylcholine/1-palmitoylphosphatidylcholine (16:0 LPC/DPPC) liposomes at gel phase. With a 16:0 LPC concentration up to 27 mol% only the sharp main transition with relatively high enthalpy (△H) values of DPPC was observed. Increasing 16 : 0 LPC concentration, the phase transition was broadened and the transition enthalpy was decreased and finally totally disappeared. The fluorescence probes of 3AS, 9AS, 12AS, and 16AP were employed, respectively, to detect the mo-bility of various sites of carbon chains of DPPC or 16:0 LPC/DPPC liposomes. It was shown that DPPC liposomes formed in the absence of 16:0 LPC always had a fluidity gradient in both gel and liquid-crystalline phase, while in the presence of 14.1 mol% and 27.0 mol% 16:0 LPC in the mixtures, the fluidity gradient tended to disappear below 40℃:     

Ln2(O2CCH2PO3)2(H2O)3 · H2O (Ln = La, Pr, Nd, Sm): Layered lanthanide phosphonoacetates containing two-dimensional -Ln-O- linkages

Four novel isostructural lanthanide phosphonate compounds with formula Ln₂(O₂CCH₂PO₃)₂(H₂O)₃ · H₂O [Ln = La (1), Pr (2), Nd (3), Sm (4)] have been prepared through hydrothermal reactions of phosphonoacetate acid and lanthanide nitrates. All show layered structures made up of {LnO₉} polyhedra and {CPO₃} tetrahedra with the lattice water molecules locating between the layers. Within the layer, chains of edge-sharing {LnO₉} polyhedra are connected via corner-sharing by phosphonate oxygens forming a two-dimensional -Ln-O- linkage. Thermal analyses and XRD measurements reveal that the framework structures can be maintained up to 400 °C.     

Crystal structures of (2R,4R)-2-(polyhydroxyalkyl)-1,3-thiazolidine-4-carboxylic acids: condensation products of l-cysteine with d-hexoses

We report herein the first crystal structures of (4-carboxy-1,3-thiazolidin-2-yl)pentitols [2-(polyhydroxyalkyl)thiazolidine-4-carboxylic acids], condensation products of l-cysteine with d-galactose and d-mannose: 2-(d-galacto-pentahydroxypentyl)thiazolidine-4-carboxylic acid hydrate, Gal-Cys·H₂O (1), and 2-(d-manno-pentahydroxypentyl)thiazolidine-4-carboxylic acid hydrate, Man-Cys·H₂O (2). In 1 and 2 the compounds crystallize as zwitterions, with the carboxylic groups deprotonated and the thiazolidine N atoms protonated. The sugar moiety and carboxylate group are in a cis configuration relative to the thiazolidinium ring, which adopts different conformation: twisted (T) on C^β–S in 1, and S-puckered envelope (E) in 2. The carbon chain of the galactosyl/mannosyl moiety remains in an extended zig-zag conformation. The orientation of the sugar O2 atom with respect to the thiazolidinium S and N atoms is trans–gauche in 1 and gauche–gauche in 2. The molecular conformation is stabilized by the intramolecular N–H?O_Cys contacts in both 1 and 2 and by the additional N–H?O_Man interaction in 2. The crystal packing of orthorhombic 1 and monoclinic 2 is determined mainly by N/O/C–H?O hydrogen bonds forming ribbons linked to each other by direct and water-mediated O/C–H?O/S contacts.     

The phase diagram of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/sucrose in the dry state. Sucrose substitution for water in lamellar mesophases

The phase diagram of the binary system, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/sucrose, was determined by DSC. In contrast to dry DPPC, which exhibits chain melting at 342.5 K, the main feature of the DPPC/sucrose system is eutectic melting at 320 K. This was supported earlier by Crowe, J.H., Crowe, L.M. and Chapman, D. (Science 223 (1984) 701-703), who reported a drastic decrease in the chain-melting temperature of the dry lipid in the presence of some mono- and disaccharides. Electron microscopy suggests that the phase structures on either side of the phase transition are of the lamellar type. Definite sugar saturation concentrations can be derived from this phase diagram. Up to about 17 mol% sucrose, i.e., 1 mol of sucrose per 5 mol of lipid is adopted by DPPC in the low-temperature phase Lc. In the high-temperature phase Lm the saturation concentration is well above 90 mol% sucrose at 320 K (eutectic point) but decreases with increasing temperature. The lower limit of 50 mol% sucrose is reached at 455 K. At this temperature, peritectic melting of sucrose occurs. Because of some similarities in the phase diagrams of DPPC/sucrose and DPPC/water, it is possible to understand the sucrose substitution for water in dry lamellar mesophases.     

Conformational difference between diastereomers of Dnp-Val-Aib-Gly-Leu-pNA studied by x-ray crystal analyses

In order to investigate the Conformational change of the α-aminoisobutyric acid (Aib) containing peptide by the D /L replacement of an amino acid residue, single crystals of two diastereomers, Dnp-L -Val-Aib-Gly-L -Leu-pNA (L -L isomer) and Dnp-D -Val-Aib-Gly-L -Leu-pNA (D -L isomer), were prepared from aqueous methanol solutions as CH₃OH and CH₃OH · H₂O solvates, respectively, and were analyzed by the x-ray diffraction method. Molecular conformation of L -L isomer adopts consecutive two different types of β-turns, a type II′ β-turn bent at Aib-Gly, and a type III β-turn bent at Gly-Leu, stabilized by two intramolecular (Leu) NH …? O?C (Val) and (pNA) NH …? O?C(Aib) hydrogen bonds. In contrast, these two intramolecular hydrogen bonds lead the D -L isomer to a distorted 3₁₀-helix conformation consisting of consecutive two type-III β-turn of Aib-Gly-Leu sequence. The most significant structural difference between these diastereomers is the mutual orientation between the Dnp and pNA chromophores. While the extensive stacking of both the chromophores is intramolecularly formed for the folded conformation of L -L isomer, they are oriented toward an opposite direction in the open conformation of D -L isomer and are intermolecularly stacked with each other. The large separation between these diastereomers observed in the chromatography is discussed in the relation with their Conformational differences. © 1993 John Wiley & Sons, Inc.     

Spectroscopic characterisation of n-octanohydroxamic acid and potassium hydrogen n-octanohydroxamate

The crystal structure and spectroscopic characteristics of n-octanohydroxamic acid and the potassium compound of that acid have been investigated by XRD, XPS, FTIR and Raman spectroscopy. XRD revealed that the acid is in the keto Z conformation with the alkyl chains oriented along the z-direction and hydrogen bonding between hydroxamate moieties. Vibrational spectra confirm this conclusion. Chemical analysis, XRD and XPS established that the potassium compound is the acid salt KH(C₇H₉CONO)₂. The crystal structure showed that the hydroxamate groups are also in the keto Z conformation and this is supported by vibrational spectra. In the acid salt, the two hydroxamate moieties are connected by a symmetrical O-H-O short hydrogen bonded linkage between the two hydroxamate oxygen atoms and this explains the absence of a discernible O-H stretch band in the vibrational spectra. Identification of the vibrational bands displayed is supported by deuteration and ¹⁵N substitution.     

Differences in hydrocarbon chain tilt between hydrated phosphatidylethanolamine and phosphatidylcholine bilayers. A molecular packing model.

Both wide-angle and lamellar x-ray diffraction data are interpreted in terms of a difference in hydrocarbon chain tilt between fully hydrated dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE). Although the hydrocarbon chains of multilayers of DPPC tilt ty approximately 30 degrees relative to the normal to the plane of the bilayer, as previously reported by others, the hydrocarbon chains of DPPE appear to be oriented approximately normal to the plane of the bilayer. It is found that the chain tilt in DPPC bilayers can be reduced by either: (a) adding an n-alkane to the bilayer interiors or (b) adding lanthanum ions to the fluid layers between bilayers. A molecular packing model is presented which accounts for these data. According to this model, DPPC chains tilt because of the size and conformation of the PC polar head group.     

In-plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids

SAXS/WAXS studies were performed in combination with freeze fracture electron microscopy using mixtures of a new Gemini catanionic surfactant (Gem16-12, formed by two sugar groups bound by a hydrocarbon spacer with 12 carbons and two 16-carbon chains) and the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) to establish the phase diagram. Gem16-12 in water forms bilayers with the same amount of hydration water as DPPC. A frozen interdigitated phase with a low hydration number is observed below room temperature. The kinetics of the formation of this crystalline phase is very slow. Above the chain melting temperature, multilayered vesicles are formed. Mixing with DPPC produces mixed bilayers above the corresponding chain melting temperature. At room temperature, partially lamellar aggregates with local nematic order are observed. Splitting of infinite lamellae into discs is linked to immiscibility in frozen state. The ordering process is always accompanied by dehydration of the system. As a consequence, an unusual order-disorder phase transition upon cooling is observed.     

The dissolution and characterization of Bombyx mori silk fibroin in calcium nitrate-methanol solution and the regeneration of films

Bombyx mori silk fibroin from the silkworm was found to be soluble in a calcium nitrate-methanol system. Fibroin dissolves in 75% w/v Ca(NO₃)₂/MeOH solution at a temperature of 67°C. The viscometric behavior of the fibroin-salt solution was analyzed and the fibroin's secondary structures were determined via ¹³C solution nmr. Fourier transform ir, solid state ¹³C-nmr, x-ray diffraction, differential scanning calorimetry, scanning electron microscopy (SEM), and polarizing microscopy were used to characterize regenerated films and fibers. A compositional phase diagram of fibroin in the salt solution was constructed. Viscosity data indicate that there is aggregation of fibroin chains within the salt solution. The extremely high value of intrinsic viscosity of 8.7 dL/g at 298 K may be due to aggregation. Aggregation may be caused by the complexing of calcium ions with the fibroin chains at their amide linkages. The energy required for viscous flow for the fibroin solution (ΔH_vis = 9.03 kcal/mol) is greater than that of the solvent (ΔH_vis = 7.01 kcal/mol). Chain entanglements may be hindering the free motion of chains thus increasing the energy required for the viscous flow. ¹³C-nmr shows that fibroin chains exist in two independent conformational environments. While most of the molecule is in a random coil conformation, there is evidence of some order within the chains of fibroin. In as-cast regenerated films, the fibroin chains are in a random coil/α-helix conformation with some β-sheet content. Crystallinity induced by immersion of thin films in methanol is evidenced via x-ray diffraction, which shows lattice spacings at 4.042 Å. Thin films have a fibrillar morphology that is clearly shown under the SEM and the polarizing microscope. Fibers were hand pulled from the concentrated fibroin-salt solutions and coagulated with acetone and methanol. A microscopic analysis was done using the polarizer. © 1997 John Wiley & Sons, Inc. Biopoly 42: 61–74, 1997     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.     

Sequence-dependence of secondary structure formation: conformational studies of host-guest peptides in alpha-helix and beta-structure supporting media

A newly designed host–guest approach is introduced as a experimental tool to explore the relationship between the sequence of peptides and their secondary structure. From the CD spectra of the host–guest peptides studied, a tentative scale for the α-helix potential in 2,2,2-trifluorethanol of guest amino acids is delineated. The conformational preferences are also examined in β-structure supporting media (solid state, CH₂Cl₂, CH₃OH, H₂O) using ir-absorption and CD techniques. Scales for the β-forming tendency of guest amino acid residues in the different media are delineated. It is shown that the preferred conformation of the host–guest peptides is a function of the medium, the chain length, and the protecting groups. Given the fact that conformational effects are important in peptide synthesis, the tentative scales may serve as a guideline to predict secondary structures of side-chain-protected or -deprotected peptides in a given solvent, complementing the well-known empirical conformational prediction parameters.     

Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

KL₄ is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL₄ binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL₄ is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL₄ is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL₄, and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL₄. Additionally, a clear effect of KL₄ on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL₄ causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL₄ to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment.     

Structure and phase behavior of hydrated mixtures of L-dipalmitoylphosphatidylcholine and palmitic acid. Correlations between structural rearrangements, specific volume changes and endothermic events

Several new features of the phase diagram of L-dipalmitoylphosphatidylcholine (DPPC)/palmitic acid mixtures in excess water were established by means of static and time-resolved X-ray diffraction, densitometry and differential scanning calorimetry (DSC). At low temperatures, palmitic acid has a biphasic effect on the lamellar subgel phases: at concentrations below 5-6 mol%, it prevents formation of the DPPC subgel phase (Lc), while at higher contents (between about 40 and 90 mol%) another subgel phase (Lccom) is formed as a result of lipid co-crystallization at 1 DPPC: 2 palmitic acid stoichiometry. A crystalline palmitic acid phase separates from Lccom above 70-80 mol% of fatty acid. The Lccomphase transforms into a lamellar gel phase (L beta) in an endothermic transition centered at 38 degrees C. At high temperatures, the mixtures form hexagonal liquid-crystalline phase (HII) in the region of 60-70 mol% and an isotropic phase (I) at 90-100 mol% of palmitic acid. No coexistence of HII phase with the fluid lamellar phase of DPPC was observed at intermediate compositions (20 and 50 mol% of palmitic acid) but rather formation of a complex phase with non-periodic geometry characterized by molten chains and a broad, continuous small-angle scattering band. No evidence for fluid phase coexistence was found also at compositions between HII and I phases. The L beta--HII transition at 60-70 mol% of palmitic acids is readily reversible and two-state in both heating and cooling modes. It is characterized by the coexistence of initial and final phases with no detectable intermediates by time-resolved and static X-ray diffraction. The crystalline-isotropic transition in palmitic acid is two-state only in heating direction. On cooling, it is characterized by strong undercooling and gradually relaxing lamellar crystalline structures. The slowly reversible Lccom--L beta transition proceeds continuously through intermediate states. Although clearly discernible by both DSC and X-ray diffraction, it is not accompanied by specific volume changes.     

Weak intramolecular interactions in perchlorate salts of Λ-β1-[Co(R,R-picchxn)(R-aa)] [picchxn=N,N-di(2-picolyl)-1,2-diaminocyclohexane; aa=phenylalaninato(1−), tyrosinato(1−)] and their diastereoisomeric equilibration in solution

Single-crystal X-ray structures and high-resolution solution NMR studies of Λ-β₁-[Co(R,R-picchxn)(R-phe)](ClO₄)₂ · H₂O and Λ-β₁-[Co(R,R-picchxn)(R-tyr)](ClO₄)₂ · 2H₂O (pheH=phenylalanine, tyrH=tyrosine) are reported. In the former, the aromatic side group of the phenylalanine ligand is extended, as is found in related halide salts. In the tyrosine analogue, the aromatic ring adopts a conformation such that a weak intramolecular NH-π interaction with a NH group of the tetradentate ligand is stabilized. The interaction is rather weak and is not particularly favoured in solution, as revealed by ¹H NMR. The β₁ complex of phenylalanine is not the thermodynamically stable diastereoisomer. Equilibration experiments give a mixture of Λ-β₁, Λ-β₂ and Δ-α diastereoisomers in D₂O solution, the α diastereoisomer being more stable than either of the β complexes by about 1 kJ mol⁻¹ at 298.2 K. Intramolecular π-π and NH-π interactions are responsible for the stabilization of the α complex, demonstrating the significance of the cooperative effects of such interactions. The ternary cobalt complexes described in this study could act as simple model systems for investigating the discriminatory effects of analogous weak interactions that occur in a complex biological setting.     

Hydrogen sulfide concentration in the milieu of the hydrated alanine dipeptide determines its polyproline II-beta propensity: Main chain contribution to the energetic origin of the formation of amyloid

In view of the observation that the concentration of hydrogen sulfide in brains with Alzheimer’s disease (AD) is lower than that in normal brains and in line with our previous studies indicating that additional content in the aqueous environment (milieu) of a peptide can change its local energetic preference from a polyproline II (P) to a β conformation (and therefore its tendency to form the β-chain structures that lead to the amyloid plaques associated with the disease), we have studied the effect of H₂S concentration on such propensity in a simple model peptide, the alanine dipeptide (ADP). The two concentration states are represented by ADP(H₂O)₁₈(H₂S) and ADP(H₂O)₁₈(H₂S)₂. Ab initio calculations of these structures show that the lowest energy of the former is a β conformation while that of the latter is a P, mirroring the observed AD results and strengthening our proposal that amyloid diseases are better viewed in the context of a protein milieu-folding paradigm.     

Intramolecular disorder and its relation to mesophase structure in lipid/water mixtures

NMR spin-half pair dipolar echo measurements are reported for the lamellar (dispersions and multibilayer stacks) and hexagonal phases of potassium palmitate/²H₂O mixtures. In the lamellar L_β and L_γ (gel) phases the alkyl chains are rigid and perfectly ordered, while in the lamellar L_α and hexagonal phases they are flexible and disordered. In particular, the measurements show that in the fluid lamellar L_α phase the chain is “bent” at the C₉–C₁₀ segment; but is “straight” in the hexagonal phase.