Cycloheximide enhances factor binding to the native 40S ribosomal subunit of Microsporum canis

Native and derived ribosomal particles from the mycelial cells of Microsporum canis grown in the presence and absence of cycloheximide were compared by CsCl equilibrium density gradient centrifugation. Since the buoyant densities of ribonucleoprotein complexes are dependent on the protein-RNA ratio, they reflect the composition of these particles. The native monosomes from cells grown in the presence and absence of cycloheximide had a buoyant density of 1.585 g/cc. The native 60S subunits showed a density of 1.540 g/cc from cells grown in both presence and absence of cycloheximide, while the derived subunits showed a density of 1.610 g/cc. The derived 40S subunits had a density of 1.550 g/cc while the native 40S showed a major species of density 1.535 g/cc with three other minor species ranging in densities from 1.450-1.390 g/cc. The mycelia grown in the presence of cycloheximide showed an increased proportion of native 40S subunits in the density range of 1.450-1.390 g/cc, indicating that the drug enhances factor binding to native ribosomal subunits in M. canis.

Common epitopes of serum retinol-binding proteins: a study with monoclonal antibodies

Monoclonal antibodies were raised against purified chicken retinol-binding protein. These were characterised extensively with respect to their ability to recognize retinol-binding proteins from different species. The monoclonal antibodies exhibited differential recognition characteristics. Though the majority presented restricted reactivities, one out of the four monoclonal antibodies studied cross-reacted with retinol-binding proteins from all species tested so far.

Double helix conformation, groove dimensions and ligand binding potential of a G/C stretch in B-DNA

The self-complementary DNA fragment CCGGCGCCGG crystallizes in the rhombohedral space group R3 with unit cell parameters a = 54.07 angstrom and c = 44.59 angstrom. The structure has been determined by X-ray diffraction methods at 2.2 angstrom resolution and refined to an R value of 16.7%. In the crystal, the decamer forms B-DNA double helices with characteristic groove dimensions: compared with B-DNA of random sequence, the minor groove is wide and deep and the major groove is rather shallow. Local base pair geometries and stacking patterns are within the range commonly observed in B-DNA crystal structures. The duplex bears no resemblance to A-form DNA as might have been expected for a sequence with only GC base pairs. The shallow major groove permits an unusual crystal packing pattern with several direct intermolecular hydrogen bonds between phosphate oxygens and cytosine amino groups. In addition, decameric duplexes form quasi-infinite double helices in the crystal by end-to-end stacking. The groove geometries and accessibilities of this molecule as observed in the crystal may be important for the mode of binding of both proteins and drug molecules to G/C stretches in DNA.

Antigenic determinants of human serum retinol binding protein as probed with monoclonal antibodies

Monoclonal antibodies raised against human serum retinol-binding protein (hRBP) were used as probes for the study of the antigenic determinants of hRBP and those shared with the same protein from other species. The antibodies could be classified into four distinct groups and react with the homologous proteins from the rat as well as the rabbit sera. Three of these antibodies recognize sequential or continuous epitopes while the remaining antibody is directed against a discontinuous or conformational epitope. By chemical cleavage with cyanogen bromide, the domains recognized by the monoclonal antibodies could be delineated. By solid-phase synthetic approach, the core sequences recognized by two of these monoclonal antibodies were identified to amino acid sequences 45–51 and 128–131 of the primary amino acid sequence of hRBP.

Computer modeling studies on the binding of 2',5'-linked dinucleoside phosphates to ribonuclease T1-influence of subsite interactions on the substrate specificity

The modes of binding of Gp(2',5')A, Gp(2',5')C, Gp(2',5')G and Gp(2',5')U to RNase T1 have been determined by computer modelling studies. All these dinucleoside phosphates assume extended conformations in the active site leading to better interactions with the enzyme. The 5'-terminal guanine of all these ligands is placed in the primary base binding site of the enzyme in an orientation similar to that of 2'-GMP in the RNase T1-2'-GMP complex. The 2'-terminal purines are placed close to the hydrophobic pocket formed by the residues Gly71, Ser72, Pro73 and Gly74 which occur in a loop region. However, the orientation of the 2'-terminal pyrimidines is different from that of 2'-terminal purines. This perhaps explains the higher binding affinity of the 2',5'-linked guanine dinucleoside phosphates with 2'-terminal purines than those with 2'-terminal pyrimidines. A comparison of the binding of the guanine dinucleoside phosphates with 2',5'- and 3',5'-linkages suggests significant differences in the ribose pucker and hydrogen bonding interactions between the catalytic residues and the bound nucleoside phosphate implying that 2',5'-linked dinucleoside phosphates may not be the ideal ligands to probe the role of the catalytic amino acid residues. A change in the amino acid sequence in the surface loop region formed by the residues Gly71 to Gly74 drastically affects the conformation of the base binding subsite, and this may account for the inactivity of the enzyme with altered sequence i.e., with Pro, Gly and Ser at positions 71 to 73 respectively. These results thus suggest that in addition to recognition and catalytic sites, interactions at the loop regions which constitute the subsite for base binding are also crucial in determining the substrate specificity.

Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes

The major heat-stable shrimp allergen (designated as Sa-II), capable of provoking IgE-mediated immediate type hypersensitivity reactions after the ingestion of cooked shrimp, has been shown to be a 34-kDa heat- stable protein containing 300 amino acid residues. Here, we report that a comparison of amino acid sequences of different peptides generated by proteolysis of Sa-II revealed an 86% homology with tropomyosin from Drosophila melanogaster, suggesting that Sa-II could be the shrimp muscle protein tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from uncooked shrimp (Penaeus indicus) and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8 to 5.4. In the presence of 6 M urea, the mobility of both Sa-II and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp-sensitive patients as assessed by competitive ELISA inhibition and Western blot analysis. Tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were superimposable. Dot-blot and competitive ELISA inhibition using sera of shrimp-sensitive patients revealed that antigenic as well as allergenic activities were associated with two peptide fractions. These IgE-binding tryptic peptides were purified and sequenced. Mouse anti-anti-idiotypic antibodies raised against Sa-II specific human idiotypic antibodies recognized not only tropomyosin but also the two allergenic peptides, thus suggesting that these peptides represent the major IgE binding epitopes of tropomyosin. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes (residues 50-66 and 153-161) with the corresponding regions of tropomyosins from different vertebrates confirmed lack of allergenic cross-reactivity between tropomyosins from phylogenetically distinct species.

Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris. Copyright (C) 2010 John Wiley & Sons, Ltd.

The Design and Construction of Synthetic Protein Mimics

A strategy for the modular construction of synthetic protein mimics based on the ability non-protein amino acids to act as stereochemical directors of polypeptide chain folding, is described. The use of alpha-aminoisobutyric acid (Aib) to construct stereochemically rigid helices has been exemplified by crystallographic and spectroscopic studies of several apolar peptides, ranging in length from seven to sixteen residues. The problem of linker design in elaborating alpha,alpha motifs has been considered. Analysis of protein crystal structure data provides a guide to choosing linking sequences. Attempts at constructing linked helical motifs using linking Gly-Pro segments have been described. The use of flexible linkers, like epsilon-aminocaproic acid has been examined and the crystallographic and solution state analysis of a linked helix motif has been presented. The use of bulky sidechain modifications on a helical scaffold, as a means of generating putative binding sites has been exemplified by a crystal structure of a peptide packed in a parallel zipper arrangement.

DNA Binding, Coprotease, and Strand Exchange Activities of Mycobacterial RecA Proteins: Implications for Functional Diversity among RecA Nucleoprotein Filaments

One of the fundamental questions concerning homologous recombination is how RecA or its homologues recognize several DNA sequences with high affinity and catalyze all the diverse biological activities. In this study, we show that the extent of single-stranded DNA binding and strand exchange (SE) promoted by mycobacterial RecA proteins with DNA substrates having various degrees of GC content was comparable with that observed for Escherichia coli RecA. However, the rate and extent of SE promoted by these recombinases showed a strong negative correlation with increasing amounts of sequence divergence embedded at random across the length of the donor strand. Conversely, a positive correlation was seen between SE efficiency and the degree of sequence divergence in the recipient duplex DNA. The extent of heteroduplex formation was not significantly affected when both the pairing partners contained various degrees of sequence divergence, although there was a moderate decrease in the case of mycobacterial RecA proteins with substrates containing larger amounts of sequence divergence. Whereas a high GC content had no discernible effect on E. coli RecA coprotease activity, a negative correlation was apparent between mycobacterial RecA proteins and GC content. We further show clear differences in the extent of SE promoted by E. coli and mycobacterial RecA proteins in the presence of a wide range of ATP:ADP ratios. Taken together, our findings disclose the existence of functional diversity among E. coli and mycobacterial RecA nucleoprotein filaments, and the milieu of sequence divergence (i.e., in the donor or recipient) exerts differential effects on heteroduplex formation, which has implications for the emergence of new genetic variants.

Effect of Substrate Binding Loop Mutations on the Structure, Kinetics, and Inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network. (C) 2011 IUBMB mum Life, 63(1): 30-41,2011

Binding of active site directed ligands to phospholipase A2: Implications on the molecular constraints and catalytic mechanism

Molecular constraints for the localization of active site directed ligands (competitive inhibitors and substrates) in the active site of phospholipase A2 (PLA2) are characterized. Structure activity relationships with known inhibitors suggest that the head : group interactions dominate the selectivity as well as a substantial part of the affinity. The ab initio fitting of the amide ligands in the active site was carried out to characterize the head group interactions. Based on a systematic coordinate space search, formamide is docked with known experimental constraints such as coordination of the carbonyl group to Ca2+ and hydrogen bond between amide nitrogen and ND1 of His48. An optimal position for a bound water molecule is identified and its significance for the catalytic mechanism is postulated. Unlike the traditional ''pseudo-triad'' mechanism, the ''Ca-coordinatedoxyanion'' mechanism proposed here invokes activation of the catalytic water to form the oxyanion in the coordination sphere of calcium. As it attacks the carbonyl carbon of the ester, a near-tetrahedral intermediate is formed. As the second proton of the catalytic water is abstracted by the ester oxygen, its reorientation and simultaneous cleavage form hydrogen bond with ND1 of His48. In this mechanism of esterolysis, a catalytic role for the water co-ordinated to Ca2+ is recognised.

Characterization of substrate UpA binding to RNase A--computer modelling and energetics approach.

In the past two decades RNase A has been the focus of diverse investigations in order to understand the nature of substrate binding and to know the mechanism of enzyme action. Although this system is reasonably well characterized from the view point of some of the binding sites, the details of interactions in the second base binding (B2) site is insufficient. Further, the nature of ligand-protein interaction is elucidated generally by studies on RNase A-substrate analog complexes (mainly with the help of X-ray crystallography). Hence, the details of interactions at atomic level arising due to substrates are inferred indirectly. In the present paper, the dinucleotide substrate UpA is fitted into the active site of RNase A Several possible substrate conformations are investigated and the binding modes have been selected based on Contact Criteria. Thus identified RNase A-UpA complexes are energy minimized in coordinate space and are analysed in terms of conformations, energetics and interactions. The best possible ligand conformations for binding to RNase A are identified by experimentally known interactions and by the energetics. Upon binding of UpA to RNase A the changes associated,with protein back bone, Side chains in general and at the binding sites in particular are described. Further, the detailed interactions between UpA and RNase A are characterized in terms of hydrogen bonds and energetics. An extensive study has helped in interpreting the diverse results obtained from a number of experiments and also in evaluating the extent of changes the protein and the substrate undergo in order to maximize their interactions.

Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.

Thermodynamics of lectin-sugar interaction: Binding of sugars to winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBAI)

Combining site of WBAI is extended and encompasses all the residues of blood group A-reactive trisaccharide [GalNAcalpha3Galbeta4Glc]. Though both of the fucose residues of A-pentasaccharide [GalNAcalpha(Fucalpha2)3Galbeta(Fucalpha3)4Glc] do not directly interact, with the combining site they thermodynamically favour the interaction of GalNAcalpha3Galbeta4Glc part of the molecule by imposing a sterically favourable orientation of the binding epitope viz. GalNAcalpha3Galbeta4Glc of the saccharide. Binding of sugars is driven by enthalpy and is devoid of heat capacity changes. This together with enthalpy-entropy compensation observed for these processes underscore the importance of water reorganization as being one of the principal determinant of protein-sugar interactions.