Anti-Group A Streptococcal Vaccine Epitope STRUCTURE, STABILITY, AND ITS ABILITY TO INTERACT WITH HLA CLASS II MOLECULES

Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue alpha-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 alpha-helix residues, whereas the B cell epitope is in the second microdomain and showed no alpha-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCoroverlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.

Gas-surface interactions in the thermal and sub-thermal regime: A molecular dynamics study

Molecular dynamics simulations are used to study the interaction of low-energy Ar atoms with the Ni(001) surface, Angular scattering distributions, in and out of the plane of incidence, are investigated as a function of incident energy, angles of incidence, crystallographic orientation of the incident beam and surface temperature. The results show a clear transition to the structure scattering regime at around 2 eV. However, at lower energies, two sub-regimes are revealed by the simulations, Far energies up to 250 meV, scattering is mainly diffuse, and significant trapping on the surface is observed, At energies above this level, lobular patterns start to form and trapping decreases with the increase in energy, Generally, there is a weak temperature dependence, but variations in the angle of incidence and/or changes in the crystallographic direction, generate significant changes in the scattering patterns.

Solution structure of the sodium channel antagonist conotoxin GS: A new molecular caliper for probing sodium channel geometry

Background: The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels. Results: The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors. Conclusions: This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

Cell-wall polysaccharides from Australian red algae of the family Solieriaceae (Gigartinales, Rhodophyta): Novel, highly pyruvated carrageenans from the genus Callophycus

Cell-wall polysaccharides from six species of red algae of the genus Callophycus were mainly galactans comprised predominantly of galactose (Gal) and 3,6-anhydrogalactose (AnGal), and were rich in pyruvate and sulfate. The Fourier Transform Infrared (FTIR) spectra of the polysaccharides superficially resembled that of alpha-carrageenan (composed of the repeating disaccharide carrabiose 2-sulfate), with major bands of absorption indicative of if-linked AnGal, axial 2-sulfate on 4-linked AnGal, and unsulfated, 3-linked Gal. The FTIR spectra of solutions of Callophycus polysaccharides in D2O-phosphate buffer displayed absorption, corresponding to the carboxylate anion of the pyruvate acetal substituent. Methylation analysis showed that 3,4,6-linked Galp (interpreted as 4,6-pyruvated, 3-linked Galp) and 2,4-linked AnGalp (interpreted as 4-linked AnGalp 2-sulfate) were the dominant links, together with significant quantities of 3-linked Galp. Proton-decoupled C-13 nuclear magnetic resonance (NMR) spectroscopy showed the polysaccharides to be composed predominantly of pyruvated carrageenans. The C-13 NMR spectra were completely assigned by a J-modulated spin-echo pulse sequence and 2D experiments employing gradient Heteronuclear Multiple Bond Correlation (HMBC), C-13/H-1 Heteronuclear Multiple Quantum Coherence (HMQC), and HMQC Total Correlation Spectroscopy (HMQC-TOCSY). The Callophycus galactans thus consist predominantly of the novel repeating disaccharide 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate and minor amounts of the alpha-carrageenan repeating unit (carrabiose 2-sulfate), and other structural variations. (C) 1997 Elsevier Science Ltd.

Structural and electron self-exchange rate variations in isomeric (hexaamine)cobalt(III/II) complexes

The syntheses and characterisation of the new macrocyclic hexaamine trans-(5(S),7(S),12(R),14(R)-tetramethyl)-1,4,8,11-tetraazacyclotetradecane-6,13-diamine (L-6) and its Co-III complex are reported. The X-ray crystal structural analyses of [CoL6]Cl-2(ClO4) [monoclinic, space group C2/c, a = 16.468(3) Angstrom, b = 9.7156(7) Angstrom, c = 15.070(3) Angstrom, beta = 119.431(8)degrees, Z = 4] and the closely related cis-diamino-substituted macrocyclic complex [CoL2](ClO4)(3) . 2H(2)O (L-2 = cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine) [orthorhombic, space group Pna2(1), a = 16.8220(8) Angstrom, b = 10.416(2) Angstrom, c = 14.219(3) Angstrom, Z = 4] reveal significant variations in the observed Co-N bond lengths and coordination geometries, which may be attributed to the trans or cis disposition of the pendent primary amines. The Co-III/II self-exchange electron transfer rate constants for these and other closely related hexaamines have been determined, and variations of some 2 orders of magnitude are found between pairs of trans and cis isomeric Co-III complexes.

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: The role of side chain rotations and cisoid/transoid interconversions

H-1 NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol(-1). This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is greater than or equal to 37 kJ mol(-1), which is significantly larger than has previously been reported for Csp(2)-Csp(3) bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.

Structure optimization combining soft-core interaction functions, the diffusion equation method, and molecular dynamics

Smoothing the potential energy surface for structure optimization is a general and commonly applied strategy. We propose a combination of soft-core potential energy functions and a variation of the diffusion equation method to smooth potential energy surfaces, which is applicable to complex systems such as protein structures; The performance of the method was demonstrated by comparison with simulated annealing using the refinement of the undecapeptide Cyclosporin A as a test case. Simulations were repeated many times using different initial conditions and structures since the methods are heuristic and results are only meaningful in a statistical sense.

Structure of galactosylononitol

A cyclitol galactoside was isolated from the seeds of Vigna angularis and shown to be identical with galactosylononitol. However, detailed 2D NMR data and methylation analysis resulted in the revision of the structure of galactosylononitol to O-alpha-D-galactopyranosyl-(1-->3)-4-O-methyl-D-myo-inositol (1). Thus, compound 1 represents the only naturally occuring methylated derivative of galactinol.

A two-dimensional fuzzy random model of soil pore structure

A new conceptual model for soil pore-solid structure is formalized. Soil pore-solid structure is proposed to comprise spatially abutting elements each with a value which is its membership to the fuzzy set ''pore,'' termed porosity. These values have a range between zero (all solid) and unity (all pore). Images are used to represent structures in which the elements are pixels and the value of each is a porosity. Two-dimensional random fields are generated by allocating each pixel a porosity by independently sampling a statistical distribution. These random fields are reorganized into other pore-solid structural types by selecting parent points which have a specified local region of influence. Pixels of larger or smaller porosity are aggregated about the parent points and within the region of interest by controlled swapping of pixels in the image. This creates local regions of homogeneity within the random field. This is similar to the process known as simulated annealing. The resulting structures are characterized using one-and two-dimensional variograms and functions describing their connectivity. A variety of examples of structures created by the model is presented and compared. Extension to three dimensions presents no theoretical difficulties and is currently under development.

Structural analysis of three His32 mutants of DsbA: Support for an electrostatic role of His32 in DsbA stability

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys3O-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of similar to 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 Variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys3O. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.

Crystal structure at 1.1 angstrom resolution of alpha-conotoxin PnIB: Comparison with alpha-conotoxins PnIA and GI

Conotoxins are small, cysteine-rich peptides isolated from the venom of Conus spp. of predatory marine snails, which selectively target specific receptors and ion channels critical to the functioning of the neuromuscular system. alpha-Conotoxins PnIA and PnIB are both 16-residue peptides (differing in sequence at only two positions) isolated from the molluscivorous snail Conus pennaceus. In contrast to the muscle-selective alpha-conotoxin GI from Conus geographus, PnIA and PnIB block the neuronal nicotinic acetylcholine receptor (nAChR). Here, we describe the crystal structure of PnIB, solved at a resolution of 1.1 Angstrom and phased using the Shake-and-Bake direct methods program. PnIB crystals are orthorhombic and belong to the space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 14.6 Angstrom, b = 26.1 Angstrom, and c = 29.2 Angstrom. The final refined structure of alpha-conotoxin PnIB includes all 16 residues plus 23 solvent molecules and has an overall R-factor of 14.7% (R-free of 15.9%). The crystal structures of the alpha-conotoxins PnIB and PnIA are solved from different crystal forms, with different solvent contents. Comparison of the structures reveals them to be very similar, showing that the unique backbone and disulfide architecture is not strongly influenced by crystal lattice constraints or solvent interactions. This finding supports the notion that this structural scaffold is a rigid support for the presentation of important functional groups. The structures of PnIB and PnIA differ in their shape and surface charge distribution from that of GI.

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and similar to 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1 --> residues or the disaccharide Galp-beta-(1 --> 2)-Galp-alpha-(1 --> linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 --> 4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating --> 4)-D-Manp-beta-(1 --> and --> Lt)-D-Glcp-beta-(1 --> branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1 --> 4)-[D-Galp-beta-(1 --> 2)-D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> 4)-D-Glcp-beta-(I --> 4)-[D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure. (C) 1997 Elsevier Science Ltd.

Targeted drug delivery to the skin and deeper tissues: Role of physiology, solute structure and disease

1. Drug delivery through the skin has been used to target the epidermis, dermis and deeper tissues and for systemic delivery, The major barrier for the transport of drugs through the skin is the stratum corneum, with most transport occurring through the intercellular region, The polarity of the intercellular region appears to be similar to butanol, with the diffusion of solutes being hindered by saturable hydrogen bonding to the polar head groups of the ceramides, fatty acids and other intercellular lipids, Accordingly, the permeability of the more lipophilic solutes is greatest from aqueous solutions, whereas polar solute permeability is favoured by hydrocarbon-based vehicles. 2. The skin is capable of metabolizing many substances and, through its microvasculature, limits the transport of most substances into regions below the dermis. 3. Although the flux of solutes through the skin should be identical for different vehicles when the solute exists as a saturated solution, the fluxes vary in accordance with the skin penetration enhancement properties of the vehicle. It is therefore desirable that the regulatory standards required for the bioequivalence of topical products include skin studies. 4. Deep tissue penetration can be related to solute protein binding, solute molecular size and dermal blood flow. 5. Iontophoresis is a promising area of skin drug delivery, especially for ionized solutes and when a rapid effect is required. 6. In general, psoriasis and other skin diseases facilitate drug delivery through the skin. 7. It is concluded that the variability in skin permeability remains an obstacle in optimizing drug delivery by this route.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 3(10) helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-l, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Comments on the Drinfeld realization of the quantum affine superalgebra U-q[gl(m backslash n)(1)] and its Hopf algebra structure

By generalizing the Reshetikhin and Semenov-Tian-Shansky construction to supersymmetric cases, we obtain the Drinfeld current realization for the quantum affine superalgebra U-q[gl(m\n)((1))]. We find a simple coproduct for the quantum current generators and establish the Hopf algebra structure of this super current algebra.