Ion channel formation by zervamicin-IIB. A molecular modelling study.

Zervamicin-IIB (Zrv-IIB) is a 16 residue peptaibol which forms voltage-activated, multiple conductance level channels in planar lipid bilayers. A molecular model of Zrv-IIB channels is presented. The structure of monomeric Zrv-IIB is based upon the crystal structure of Zervamicin-Leu. The helical backbone is kinked by a hydroxyproline residue at position 10. Zrv-IIB channels are modelled as helix bundles of from 4 to 8 parallel helices surrounding a central pore. The monomers are packed with their C-terminal helical segments in close contact, and the bundles are stabilized by hydrogen bonds between glutamine 11 and hydroxyproline 10 of adjacent helices. Interaction energy profiles for movement of three different probes species (K+, Cl- and water) through the central pore are analyzed. The conformations of: (a) the sidechain of glutamine 3; (b) the hydroxyl group of hydroxyproline 10; and (c) the C-terminal hydroxyl group are "optimized" in order to maximize favourable interactions between the channel and the probes, resulting in favourable interaction energy profiles for all three. This suggests that conformational flexibility of polar sidechains enables the channel lining to mimic an aqueous environment.

Unfolding of Plasmodium falciparum Triosephosphate Isomerase in Urea and Guanidinium Chloride: Evidence for a Novel Disulfide Exchange Reaction in a Covalently Cross-Linked Mutant†

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The C-m(urea)/C-m(GdmCl) ratio (where C-m is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide crosslinked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Crystal structure of peanut lectin, a protein with an unusual quaternary structure

The x-ray crystal structure of the tetrameric T-antigen-binding lectin from peanut, M(r) 110,000, has been determined by using the multiple isomorphous replacement method and refined to an R value of 0.218 for 22,155 reflections within the 10- to 2.95-A resolution range. Each subunit has essentially the same characteristic tertiary fold that is found in other legume lectins. The structure, however, exhibits an unusual quaternary arrangement of subunits. Unlike other well-characterized tetrameric proteins with identical subunits, peanut lectin has neither 222 (D2) nor fourfold (C4) symmetry. A noncrystallographic twofold axis relates two halves of the molecule. The two monomers in each half are related by a local twofold axis. The mutual disposition of the axes is such that they do not lead to a closed point group. Furthermore, the structure of peanut lectin demonstrates that differences in subunit arrangement in legume lectins could be due to factors intrinsic to the protein molecule and, contrary to earlier suggestions, are not necessarily caused by interactions involving covalently linked sugar. The structure provides a useful framework for exploring the structural basis and the functional implications of the variability in the subunit arrangement in legume lectins despite all of them having nearly the same subunit structure, and also for investigating the general problem of "open" quaternary assembly in oligomeric proteins.

Thermodynamics of monosaccharide binding to concanavalin A, pea (Pisum sativum) lectin, and lentil (Lens culinaris) lectin

Titration calorimetry measurements of the binding of methyl alpha-D-mannopyranoside (Me alpha Man), D-mannopyranoside (Man), methyl alpha-D-glucopyranoside (Me alpha Glu), and D-glucopyranoside (Glu) to concanavalin A (Con A), pea lectin, and lentil lectin were performed at 281 and 292 K in 0.01 M dimethylglutaric acid-NaOH buffer (pH 6.9) containing 0.15 M NaCl and Mn+2 and Ca+2 ions. The site binding enthalpies, delta H, are the same at both temperatures and range from -28.4 +/- 0.9 (Me alpha Man) to -16.6 +/- 0.5 kJ mol-1 (Glu) for Con A, from -26.2 +/- 1.1 (Me alpha Man) to -12.8 +/- 0.4 kJ mol-1 (Me alpha Glu) for pea lectin, and from -16.6 +/- 0.7 (Me alpha Man) to -8.0 +/- 0.2 kJ mol-1 (Me alpha Glu) for lentil lectin. The site binding constants range from 17 +/- 1 x 10(3) M-1 (Me alpha Man to Con A at 281.2 K) to 230 +/- 20 M-1 (Glu to lentil lectin at 292.6 K) and exhibit high specificity for Con A where they are in the Me alpha Man:Man:Me alpha Glu:Glu ratio of 21:4:5:1, while the corresponding ratio is 5:2:1.5:1 for pea lectin and 4:2:2:1 for lentil lectin. The higher specificity for Con A indicates more interactions between the amino acid residues at the binding site and the carbohydrate ligand than for the pea and lentil lectin-carbohydrate complexes. The carbohydrate-lectin binding results exhibit enthalpy-entropy compensation in that delta Hb (kJ mol-1) = -1.67 +/- 0.06 x 10(4) + (1.30 +/- 0.12)T(K) delta Sb (J mol-1K-1). Differential scanning calorimetry measurements on the thermal denaturation of the lectins and their carbohydrate complexes show that the Con A tetramer dissociates into monomers, while the pea and lentil lectin dimers dissociate into two submonomer fragments. At the denaturation temperature, one carbohydrate binds to each monomer of Con A and the pea and lentil lectins. Complexation with the carbohydrate increases the denaturation temperature of the lectin and the magnitude of the increases yield binding constants in agreement with the determinations from titration calorimetry.

Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus)

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.

Protein sequence design on the basis of topology optimization techniques -Using continuous modeling of discrete amino acid types

The notion of optimization is inherent in protein design. A long linear chain of twenty types of amino acid residues are known to fold to a 3-D conformation that minimizes the combined inter-residue energy interactions. There are two distinct protein design problems, viz. predicting the folded structure from a given sequence of amino acid monomers (folding problem) and determining a sequence for a given folded structure (inverse folding problem). These two problems have much similarity to engineering structural analysis and structural optimization problems respectively. In the folding problem, a protein chain with a given sequence folds to a conformation, called a native state, which has a unique global minimum energy value when compared to all other unfolded conformations. This involves a search in the conformation space. This is somewhat akin to the principle of minimum potential energy that determines the deformed static equilibrium configuration of an elastic structure of given topology, shape, and size that is subjected to certain boundary conditions. In the inverse-folding problem, one has to design a sequence with some objectives (having a specific feature of the folded structure, docking with another protein, etc.) and constraints (sequence being fixed in some portion, a particular composition of amino acid types, etc.) while obtaining a sequence that would fold to the desired conformation satisfying the criteria of folding. This requires a search in the sequence space. This is similar to structural optimization in the design-variable space wherein a certain feature of structural response is optimized subject to some constraints while satisfying the governing static or dynamic equilibrium equations. Based on this similarity, in this work we apply the topology optimization methods to protein design, discuss modeling issues and present some initial results.

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.

Anti-amyloidogenic property of leaf aqueous extract of Caesalpinia crista

Amyloid beta (A beta) is the major etiological factor implicated in Alzheimer's disease (AD). A beta(42) self-assembles to form oligomers and fibrils via multiple aggregation process. The recent studies aimed to decrease A beta levels or prevention of A beta aggregation which are the major targets for therapeutic intervention. Natural products as alternatives for AD drug discovery are a current trend. We evidenced that Caesalpinia crista leaf aqueous extract has anti-amyloidogenic potential. The studies on pharmacological properties of C. crista are very limited. Our study focused on ability of C. crista leaf aqueous extract on the prevention of (i) the formation of oligomers and aggregates from monomers (Phase I: A beta(42) + extract co-incubation); (ii) the formation of fibrils from oligomers (Phase II: extract added after oligomers formation); and (iii) dis-aggregation of pre-formedfibrils (Phase III: aqueous extract added to matured fibrils and incubated for 9 days). The aggregation kinetics was monitored using thioflavin-T assay and transmission electron microscopy (TEM). The results showed that C. crista aqueous extract could able to inhibit the A beta(42) aggregation from monomers and oligomers and also able todis-aggregate the pre-formed fibrils. The study provides an insight on finding new natural products for AD therapeutics. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Synthesis of Poly(1,4-Divinylbenzene Peroxide)

Polymeric peroxides have received renewed attention in the recent past, in view of some significant explorations of their physical and chemical properties. The potential of polymeric peroxides as a class, as specialized fuel, and the need to synthesize such new materials have been reported in the literature. So far, this class of polymers is constituted only by a dozen or so polyperoxides. From the point of view of their use in propellant applications, the importance lies in making materials which are easy to handle etc., unlike the earlier reported poly(styrene peroxide) (PSP), a sticky semi-solid mass. However, judging from the better combustion characteristics, exploring aromatic monomers was thought worthwhile. In this preliminary communication, the synthesis of a new polymeric peroxide made from 1,4-divinylbenzene is reported. The polymer obtained was in powder form and had an exothermic heat of degradation approximately equal to that of PSP. 4 ref.--AA

X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 A resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

Unfolding of Plasmodium falciparum triosephosphate isomerase in urea and guanidinium chloride solutions. Evidence for a novel disulfide exchange reaction in a covalently crosslinked mutant

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74‘) and (13‘-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol−disulfide exchange.

Physicochemical behaviour of polyperoxide

Polymeric peroxides are equimolar alternating copolymers formed by the reaction of vinyl monomers with oxygen. Physicochemical studies on the microstructure and chain dynamics of poly(styrene peroxide) PSP were first carried out by Cais and Bovey. We have found that polyperoxides are formed as main intermediates in solid-propellant combustion by the interaction of the monomer and oxygen generated by the decomposition of the polymeric binder and the oxidizer ammonium perchlorate. The experimentally determined heat of degradation and that calculated from thermochemical considerations reveal that polyperoxides undergo highly exothermic primary degradation, the rate-controlling step being the O-O bond dissociation. A random-chain scission mechanism for the thermal degradation of polyperoxides has been proposed. The prediction of unusual exothermic degradation of polyperoxides has resulted in the discovery of an interesting new phenomenon of 'autopyrolysability' in polymers. Several new polyperoxides based on vinyl naphthalene have been synthesized. We have also found that PSP, in conjunction with amines, can be used as initiator at ambient temperature for the radical polymerization of vinyl monomers.

A simple n-state model for water

A simple n-state configurational excitation model which takes into account the presence of weakly connected pentamer units in liquid water is proposed. The model has features of both the “continuum” and “mixture” models. Calculations based on this model satisfactorily account for the important, diagnostic thermodynamic properties of water such as the density maximum, fraction of monomers and so on.