Theoretical studies on penicillins, penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues: binding specificities of transeptidases and penicillinases

Empirical potential energy calculations have been carried out to determine the preferred conformations of penicillins and penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues. With the exception of 1-oxa-1-dethia penicillins, all the other compounds favour C2 and the C3 puckered conformations of their five-membered rings. Replacement of C2 methyl groups by hydrogen atoms as in bisnorpenicillin V or oxidation of sulphur in position 1 as in sulphones, makes the C3 puckered form much less favourable. Addition of an amino-acyl group at the C6 atom, however, makes the C3 puckered form more favoured in penicillin G or V and in 1-carba-1-dethia penicillins. Through the replacement of the sulphur atom at position 1 by an oxygen atom or by a -CH2 group increases the non-planarity of the lactam peptide bond, it significantly affects the relative disposition of the C3 carboxyl group with respect to the β-lactam ring. These conformational differences have been correlated with the biological activities of these compounds. The present study suggests that the conformation of the bicyclic ring system may be more important for initial binding with the crosslinking enzyme(s) involved in the biosynthesis of bacterial cell-wall peptidoglycan and that the mode of binding is influenced by the nature of the side-group at the C6 atom. These studies predict, in agreement with experimental results, that the 1-oxa-1-dethia penicillin nulceus is an inhibitor of penicillianses. The study also suggests that the stereospecificities of the crosslinking enzyme(s) and penicillinases are very similar with regard to the nature of the side-group at the 6 atom and the confirmation of the bicyclic ring system. However, the confirmational requirement for the bicyclic ring system appears to be more specific in the former enzyme than in the latter.

An essential arginine residue at the substrate binding site of 4-hydroxyisophthalate hydroxylase.

4-Hydroxyisophthalate hydroxylase was inactivated by treatment with phenylglyoxal by a process obeying pseudo-first order kinetics indicating the presence of an essential arginine located presumably in the active site. Addition of saturating amounts of 4-hydroxyisophthalate during the treatment resulted in complete protection of the enzyme from the inactivation, but addition of NADPH was totally ineffective. Analysis of the effect of various substrate analogs on the protection of the enzyme showed that carboxyl and hydroxyl groups at para positions on the aromatic ring are essential for substrate binding to the active site. It was also observed that analogs which protect the enzyme against phenylglyoxal inactivation are themselves effective inhibitors of the enzyme activity.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to a canonical LAGLIDADG homing endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches including four complementary footprinting assays such as DNase I, Cu/phenanthroline, methylation protection and KMnO4, enhancement of 2-aminopurine fluorescence and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site and generates two staggered double-strand breaks. Taken together, these results implicate that PI-MleI possess a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of LAGLIDADG family of homing endonucleases

Nature of the thiamin-binding protein from chicken egg yolk

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.

Studies on bicuculline binding sites on neuronal membrane using fluorescent antibody technique: Comparative binding of gaba and bicuculline

Highly purified fluorescent labelled anti-bicuculline antibodies were used to mark bicuculline binding sites in cerebral cortex of monkey brain. Specific binding of bicuculline could be demonstrated in the synaptosomal fraction, when bicuculline was added both Image and Image . Addition of γ-aminobutyric acid (GABA) to the bicucullinised membrane led to a decrease in fluorescence indicating same receptor loci and establishing GABA-bicuculline antagonism at a molecular level.

Estrogen-induced synthesis of riboflavin-binding protein in immature chicks kinetics and hormonal specificity

The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay proceudre. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h. reaching peak levels around 48 h and declining thereafter. A two-fold amplication of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulat ions with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered 125I-labelled protein. Simultaneous administration of progestrone did bot affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively countered the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the anti-esterogens per se were completely ineffective in substituting for estrogen in the inductive ptrocess.

Chemical modification studies on Abrus agglutinin Involvement of tryptophan residues in sugar binding

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydratebinding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2- hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

Oestrogen induction of riboflavin-binding protein in immature chicks. Nature of the secretory protein.

The riboflavin-binding protein isolated from sera of oestrogen-treated male chicks as well as that synthesized and secreted in vitro by the chicken liver have the same molecular size as that of the egg-yolk protein. Functionally the serum and yolk proteins are similar. This is in contrast with the hormone-induced synthesis, secretion and deposition of phosvitin and lipovitellin in the ovary.

New ternary copper(II) complexes of L-alanine and heterocyclic bases: DNA binding and oxidative DNA cleavage activity

Four new ternary copper(II) complexes of alpha-amino acid having polypyridyl bases of general formulation [Cu(L-ala)(B)(H2O)](X)(1-4), where L-ala is L-alanine, B is an N,N-donor heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2) and 5,6-phenanthroline dione (dione, 3), dipyrido[3,2:2',3'-f] quinoxaline (dpq, 4), and X = ClO4-/NO3- are synthesized, characterized by various spectroscopic and X-ray crystallographic methods. The complexes show a distorted square-pyramidal (4 + 1) CuN3O2 coordination geometry. The one-electron paramagnetic complexes (1-4) display a low energy d-d band near 600 nm in aqueous medium and show a quasi-reversible cyclic voltammetric response due to one-electron Cu(II)/Cu(I) reduction near - 100 mV (versus SCE) in DMF-0.1 M TBAP. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. All the complexes barring the complexes 1 and 3 are avid binder to the CT-DNA in the DNA minor groove giving an order: 4 > 2 >>>1, 3. The complexes 2 and 4 show appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent. Hydroxyl radical was investigated to be the DNA cleavage active species. Control experiments in the presence of distamycin-A show primarily minor groove-binding propensity for the complexes 2 and 4 to the DNA.

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 Å resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is dose to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.

Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica

The thermodynamics of tie binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein, The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium, Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites, These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCI in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

A common epitope of beta-lactoglobulin and serum retinol-binding proteins: Elucidation of its core sequence using synthetic peptides

One of the monoclonal antibodies raised against bovine beta-lactoglobulin reacted with human serum retinol binding protein. The finding that this monoclonal antibody also reacted with the serum retinol binding proteins isolated from other animals, suggested that this epitopic conformation is conserved among these proteins. Using ELISA and various synthetic peptides of defined sequence, we show in this paper that the epitope defined by this monoclonal antibody comprises of the highly conserved core sequence of DTDY present in beta-lactoglobulin and retinol binding proteins.