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.

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.

Study of thermal fluctuations during thermal denaturation of DNA

In this paper we address the fundamental issue of temperature fluctuation during the thermal denaturation (or the unzipping of the two strands on heating) of double stranded (ds) DNA. From our experiments we observe the presence of extremely high thermal fluctuations during DNA denaturation. This thermal fluctuation is several orders higher than the thermal fluctuation at temperatures away from the denaturation temperature range. This fluctuation is absent in single stranded (ss) DNA. The magnitude of fluctuation is much higher in heteropolymeric DNA and is almost absent in short homopolymeric DNA fragments. The temperature range over which the denaturation occurs (i.e., over which the thermal fluctuation is large) depends on the length of the DNA and is largest for the longest DNA.

A Comparative Study of Aromaticity in Substituted Tetracyclic and Hexacyclic Thiophenes

We have studied the nature of aromaticity in expanded porphyrinic analogues of thiophenes formed by four and six thiophenes. Using density functional theory (DFT) we have analyzed the aromaticity of both the macrocycle and individual molecular fragments. We find paramagnetic annulenic ring currents in the case of tetracyclic molecules and diamagnetic annulenic ring currents for hexacyclic molecules. We have also studied the effect of substitution of benzene rings within the macrocycle. We find that as the number of benzene rings is increased the aromaticity increases for tetracyclic systems and decreases for hexacyclic systems. All the results have been analyzed with various microscopic parameters, including geometry, excitation gap, and NMR criteria.

An Integrated Model of Designing

Current design models and frameworks describe various overlapping fragments of designing. However, little effort exists in consolidating these fragments into an integrated model. We propose a model of designing that integrates product and process facets of designing by combining activities, outcomes, requirements, and solutions. Validation of the model using video protocols of design sessions demonstrates that all the constructs are used naturally by designers but often not to the expected level, which hinders the variety and resulting novelty of the concepts developed in these sessions. To resolve this, a prescriptive framework for supporting design for variety and novelty is proposed and plans for its implementation are created. DOI: 10.1115/1.3467011]

Transferability of Multipole Charge Density Parameters for Supramolecular Synthons: A New Tool for Quantitative Crystal Engineering

The modularity of the supramolecular synthon is used to obtain transferability of charge density derived multipolar parameters for structural fragments, thus creating an opportunity to derive charge density maps for new compounds. On the basis of high resolution X-ray diffraction data obtained at 100 K for three compounds methoxybenzoic acid, acetanilide, and 4-methyl-benzoic acid, multipole parameters for O-H center dot center dot center dot O carboxylic acid dimer and N-H center dot center dot center dot O amide infinite chain synthon fragments have been derived. The robustness associated with these supramolecular synthons has been used to model charge density derived multipolar parameters for 4-(acetylamino)benzoic acid and 4-methylacetanilide. The study provides pointers to the design and fabrication of a synthon library of high resolution X-ray diffraction data sets. It has been demonstrated that the derived charge density features can be exploited in both intra- and intermolecular space for any organic compound based on transferability of multipole parameters. The supramolecular synthon based fragments approach (SBFA) has been compared with experimental charge density data to check the reliability of use of this methodology for transferring charge density derived multipole parameters.

Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing

Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 ( glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the ``intron'' regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing.

Characterization of the zinc-metalloprotein nature of rat spermatidal protein TP2

Spermatidal transition protein, TP2, was purified from rat testes by Hg-affinity chromatography. The present study reports the details of the zinc-metalloprotein nature of TP2 by employing the Zn-65-blotting technique. Chemical modification of cysteine by iodoacetic acid, and histidine by diethylpyrocarbonate, resulted in a near complete inhibition of Zn-65-binding to TP2. The (65)Zinc-binding was localized to the V8 protease-derived N-terminal two-third polypeptide fragment. Circular dichroism spectroscopy studies of TP2 (zinc pre-incubated) and its V8 protease-derived polypeptide fragments revealed that the N-terminal fragment has a Type I-beta-turn spectrum, while the C-terminal fragment has a small but significant alpha-helical structure. EDTA altered the circular dichroism spectrum of TP2 and the N-terminal fragment (zinc binding domain) but not that of the C-terminal fragment.

Conformationally Restricted Criegee Intermediates: Evidence for Formation and Stereoelectronically Controlled Fragmentation

The Baeyer-Villiger reaction of 2-(2-oxocyclohexyl) acetic acid occurs via a bicyclic Criegee intermediate, which fragments with stereoelectronic control, as evidenced by product analysis; the reaction of the but-2-yl ester and of 2-(2-oxocyclopentyl) acetic acid also show evidence of such stereoelectronic control, but less convincingly.

Excision of uracil from the ends of double stranded DNA by uracil DNA glycosylase and its use in high efficiency cloning of PCR products

We show that uracil DNA glycosylase from E. coli excises uracil residues from the ends of double stranded oligos. This information has allowed us to develop an efficient method of cloning PCR amplified DNA. In this report, we describe use of this method in cloning of E. coli genes for lysyl- and methionyl-tRNA synthetases. Efficiency of cloning by this method was found to be the same as that of subcloning of DNA restriction fragments from one vector to the other vector. Possibilities of using other DNA glycosylases for such applications are discussed.

Electrical switching studies on Ge-Te-Tl chalcogenide glasses: Effect of thallium on the composition dependence of switching voltages

Bulk Ge(17)Te83_,JI glasses (05x.5_13), have been found to exhibit memory type electrical switching. The switching voltages (also known as threshold voltage V-th) of Ge17Te83-xTlx glasses are found to decrease with increasing thallium content. The rate of decrease of Vtry is greater at lower concentrations and \textbackslashid, falls at a slower rate for higher thallium concentrations (x 6). The addition of thallium to the Ge-Te network fragments the covalent network and introduces ionic nature to it; the reduction in network connectivity leads to the decrease in switching voltages with thallium content. The decrease in the glass transition temperatures of Ge17Te83-xTlx glasses with increasing thallium concentration supports the idea of decrease in network connectivity with TI addition. The more metallic nature of TI also contributes to the observed reduction in the switching voltages of Ge17Te83-xTlx glasses with TI content. Further, there is an interesting correlation seen between the threshold voltage V-th and the average bond energy, as a function of TI content. In addition, the switching voltages of Ge17Te83-xTlx glasses have been found to decrease with sample thickness almost linearly. The set-reset studies indicate that the Ge17Te83-xTl2 sample can be switched for more than 10 cycles, whereas other glasses could not be reset beyond two switching cycles. (C) 2010 Elsevier B.V. All rights reserved.

Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the M13Nle mutation in ribonuclease-S

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex. We have substituted the wild-type residue at position 13, methionine (Met), with norleucine (Nle), where the only covalent change is the replacement of the sulfur atom with a methylene group. The thermodynamic parameters associated with the binding of this variant to S-protein, determined by titration calorimetry in the temperature range 10-40 degrees C, are reported and compared to values previously reported [Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426] for other position 13 analogs. The differences in the free energy and enthalpy of binding between the Met and Nle peptides are 0.6 and 7.9 kcal/mol at 25 degrees C, respectively. These differences are slightly larger than, but comparable to, the differences in the values for the Met/Ile and Met/Leu pairs. The structure of the mutant complex was determined to 1.85 Angstrom resolution and refined to an R-factor of 17.4% The structures of mutant and wild-type complexes are practically identical although the Nle side chain has a significantly higher average B-factor than the corresponding Met side chain. In contrast, the B-factors of the atoms of the cage of residues surrounding position 13 are all somewhat lower in the Nle variant than in the Met wild-type. Thus, the large differences in the binding enthalpy appear to reside entirely in the difference in chemical properties or dynamic behavior of the -S- and -CH2- groups and not in differences in the geometry of the side chains or the internal cavity surface. In addition, a novel method of obtaining protein stability data by means of isothermal titration calorimetry is introduced.

Topology of wolfgram proteins and 2?, 3?-cyclic nucleotide 3?-phosphodiesterase in CNS myelin: Studies with proteases

The topological disposition of Wolfgram proteins (WP) and their relationship with 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W > 14KDa were detected on the gels. Therefore protein fragments of WP with M.W < 14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis.

Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.

Cloning And Characterization of Mycobacteriophage-13 Promoters

Restriction fragments of mycobacteriophage 13 DNA capable of initiating transcription have been cloned into a promoter selection vector of Escherichia coli, and selected on the basis of development of resistance to chloramphenicol. The growth pattern of these 'promoter clones' on a concentration gradient of chloramphenicol and the biochemical assays of the chloramphenicol acetyl transferase have permitted the assessment of their relative promoter strengths. DNA sequence analysis revealed significant homology of these promoters to the -35 regions of the mycobacterial- and E. coli promoter consensus, but less so to the - 10 region. Based on the sequence of phage 13 promoters identified here and the reported sequences of mycobacterial promoters, a promoter consensus for mycobacteria has been generated.