Theoretical-Studies on the Modes of Binding of Some of the Beta-Glycosidically Linked Glucobioses to Concanavalin-A

The probable modes of binding for methyl-α-d-sophoroside, methyl-β-d-sophoroside, laminariboise and cellobiose to concanavalin A have been determined using theoretical methods. Methyl-d-sophorosides can bind to concanavalin A in two modes, i.e. by placing their reducing as well as non-reducing sugar units in the carbohydrate specific binding site, whereas laminaribiose and cellobiose can reach the binding site only with their non-reducing glucose units. However, the probability for methyl-α-d-sophoroside to bind to concanavalin A with its reducing sugar residue as the occupant of the binding site is much higher than it is with its non-reducing sugar residue as the occupant of the sugar binding site. A few of the probable conformers of methyl-β-d-sophoroside can bind to concanavalin A with either the reducing or non-reducing glucose unit. Higher energy conformers of cellobiose or laminaribiose can reach the binding site with their non-reducing residues alone. The relative differences in the binding affinities of these disaccharides are mainly due to the differences in the availability of proper conformers which can reach the binding site and to non-covalent interactions between the sugar and the protein. This study also suggests that though the sugar binding site of concanavalin A accommodates a single sugar residue, the residue outwards from the binding site also interacts with concanavalin A, indicating the existence of extended concanavalin A carbohydrate interactions.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.

Evidence for the inability of the intestinal mitochondria of the silkworm, Bombyx mori L.to oxidize fatty acids

Conditions for the preparation of mitochondria from silkworm intestines have been standardized. The inability of mitochondria to oxidize fatty acids has been demonstrated. Evidence for the absence of an inhibitor in the mitochondria has been obtained.

The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins

Formylation of the initiator tRNA is essential for normal growth of Escherichia coil, The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon, However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation, In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient, The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms.

Conformational changes of the chaperone SecB upon binding to a model substrate - bovine pancreatic trypsin inhibitor (BPTI)

SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.

Extension of the universal erosive burning law to partly symmetric propellant grain geometries

This paper aims at extending the universal erosive burning law developed by two of the present authors from axi-symmetric internally burning grains to partly symmetric burning grains. This extension revolves around three dimensional flow calculations inside highly loaded grain geometry and benefiting from an observation that the flow gradients normal to the surface in such geometries have a smooth behavior along the perimeter of the grain. These are used to help identify the diameter that gives the same perimeter the characteristic dimension rather than a mean hydraulic diameter chosen earlier. The predictions of highly loaded grains from the newly chosen dimension in the erosive burning law show better comparison with measured pressure-time curves while those with mean hydraulic diameter definitely over-predict the pressures. (c) 2013 IAA. Published by Elsevier Ltd. All rights reserved.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the omega Subunit

The widely conserved omega subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of omega is known to be substituted by GroEL in omega-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of omega variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to omega, which is largely unstructured, omega mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, omega(6) bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that omega(6) binds to beta ` subunit in vitro with greater affinity than that of omega. The reconstituted RNAP holoenzyme in the presence of omega(6) in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant omega results in death of the cell. Furthermore, lethality of omega(6) is relieved in cells expressing the rpoC2112 allele encoding beta ` (2112), a variant beta ` bearing Y457S substitution, immediately adjacent to the beta ` catalytic center. Our results suggest that the enhanced omega(6)-beta ` interaction may perturb the plasticity of the RNAP active center, implicating a role for omega and its flexible state.

USEFUL EXTENSIONS OF THE HENRY REACTION: EXPEDITIOUS ROUTES TO NITROALKANES AND NITROALKENES IN AQUEOUS MEDIA

The products of the Henry nitroaldol reaction from nitromethane and several aldehydes were reduced to the corresponding nitroalkanes with (n-Bu)(3)SnH in water under microwave irradiation (80 degrees C/10 min), or dehydrated to the corresponding nitroalkenes with K2CO3 in water (generally 0-5 degrees C/20 min). Both ``one-pot'' reactions occur in excellent yields across a range of aliphatic and aromatic (including heteroaromatic) substrates. It seems likely that the deoxygenation of the nitroaldols occurs via coordination of an oxygen atom of the nitro group with a tin atom, which facilitates hydride delivery in the transition state. The elimination of water from the nitroaldols in mild base is likely driven by the stability of the conjugated nitroalkene products. The elimination required workup with 2N HCl, which likely displaces a nitroalkane-nitroalkene equilibrium towards the latter. These extensions of the Henry reaction lead to products not easily obtained otherwise.