Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

Helix termination and chain reversal: Crystal and molecular structure of the alpha,beta-dehydrooctapeptide Boc-Val Delta Phe-Phe-Ala-Leu-Ala-Delta Phe-Leu-OH

The crystal structure of the dehydro octapeptide Boc-Val-Delta Phe-Phe-Ala-Leu-Ala-Delta Phe-Leu-OH has been determined to atomic resolution by X-ray crystallographic methods. The crystals grown by slow evaporation of peptide solution in methanol/water are orthorhombic, space group P2(1)2(1)2(1). The unit cell parameters are a = 8.404(3), b = 25.598(2) and c = 27.946(3) Angstrom, Z = 4. The agreement factor is R = 7.58% for 3636 reflections having (\F-o\) greater than or equal to 3 sigma (\F-o\). The peptide molecule is characterised by a 3(10)-helix at the N-terminus and a pi-turn at the C-terminus. This conformation is exactly similar to the helix termination features observed in proteins. The pi-turn conformation observed in the octapeptide is in good agreement with the conformational features of pi-turns seen in some proteins. The alpha(L)-position in the pi-turn of the octapeptide is occupied by Delta Phe(7), which shows that even bulky residues can be accommodated in this position of the pi-turns. In proteins, it is generally seen that alpha(L)-position is occupied by glycine residue. No intermolecular head-to-tail hydrogen bonds are observed in solid state structure of the octapeptide. A water molecule located in the unit cell of the peptide molecule is mainly used to hold the peptide molecule together in the crystal. The conformation observed for the octapeptide might be useful to understand the helix termination and chain reversal in proteins and to construct helix terminators for denovo protein design.

Influence of glycosidic linkage on the nature of carbohydrate binding in beta-prism I fold lectins: An X-ray and molecular dynamics investigation on banana lectin-carbohydrate complexes

The three crystal structures reported here provide details of the interactions of mannose and the mannosyl-alpha-1,3-mannose component of a pentamannose with banana lectin and evidence for the binding of glucosyl-alpha-1,2-glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-beta-1,3-glucose. Modeling studies on the three disaccharide complexes with the reducing end and the nonreducing end at the primary binding site are also provided here. The results of the Xray and modeling studies show that the disaccharides with an alpha-1,3 linkage prefer to have the nonreducing end at the primary binding site, whereas the reducing end is preferred at the site when the linkage is beta-1,3 in mannose/glucose-specific beta-prism I fold lectins. In the corresponding galactose-specific lectins, however, alpha-1,3-linked disaccharides cannot bind the lectin with the nonreducing end at the primary binding site on account of steric clashes with an aromatic residue that occurs only when the lectin is galactose-specific. Molecular dynamics simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin.

Crystal and molecular structure of Boc-Phe-Val-OMe; comparison of the peptide conformation with its dehydro analogue

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P2(1)2(1)2(1), a = 11.843(2), b = 21.493(4), c = 26.676(4)Angstrom and V = 6790 Angstrom(3). Data were collected on a CAD4 diffractometer using MoK2 radiation (lambda = 0.7107 Angstrom) up to Bragg angle theta = 26 degrees. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystallographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val(2) residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-Delta Phe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-Delta Phe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the Delta Phe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-Delta Phe-Val-OH are conformationally different, they exhibit similar packing patterns in the solid state. (C) Munksgaard 1995.

Reactivities of aluminium and aluminium-magnesium alloy powders in polymeric composites

Polymeric compositions containing Al-Mg alloys show higher reactivities, in comparison with similar compositions containing aluminium. This is observed irrespective of the amount of oxidizer, type of oxidizer used, type of polymeric binder, and over a range of the particle sizes of the metal additive. This is evident from the higher calorimetric values obtained for compositions containing the alloy, in comparison to samples containing aluminium. Analysis of the combustion residue shows the increase in calorimetric value to be due to the greater extent of oxidation of the alloy. The interaction between the polymeric binder and the alloy was studied by coating the metal particles with the polymer by a coacervation technique. On ageing in the presence of ammonium perchlorate, cracking of the polymer coating on the alloy was noticed. This was deduced from differential thermal analysis experiments, and confirmed by scanning electron microscopic observations. The increase in stiffness of the coating, leading to cracking, has been traced to the cross-linking of the polymer by magnesium.

The stability of transmembrane helices: A molecular dynamics study on the isolated helices of bacteriorhodopsin

Bacteriorhodopsin (bR) continues to be a proven testing ground for the study of integral membrane proteins (IMPs). It is important to study the stability of the individual helices of bR, as they are postulated to exist as independently stable transmembmne helices (TMHs) and also for their utility as templates for modeling other IMPs with the postulated seven-helix bundle topology. Toward this purpose, the seven helices of bR have been studied by molecular dynamics simulation in this study. The suitability of using the backbone-dependent rotamer library of side-chain conformations arrived at from the data base of globular protein structures in the case TMHs has been tested by another set of ? helix simulations with the side-chain orientations taken from this library. The influence of the residue's net charge oil the helix stability was examined by simulating the helices III, IV, and VI (from both of the above sets of helices) with zero net charge on the side chains. The results of these 20 simulations demonstrate in general the stability of the isolated helices of bR in conformity with the two-stage hypothesis of IMP folding. However, the helices I, II, V, and VII are more stable than the other three helices. The helical nature of certain regions of III, IV, and VI are influenced by factors such as the net charge and orientation of several residues. It is seen that the residues Arg, Lys, Asp, and Glu (charged residues), and Ser, Thr, Gly, and Pro, play a crucial role in the stability of the helices of bR. The backbone-dependent rotamer library for the side chains is found to be suitable for the study of TMHs in IMP. (C) 1996 John Wiley & Sons, Inc.

The unusual formation of methyl alpha-(5,6-dimethoxycarbonyl-2,3-dimethoxyazepin-7-ylidene)-alpha-[5-methoxycarbonyl-2,3-dimethoxypyrid-6-yl)acetate during the pyrolysis of ''azido-meta-hemipinate'' - First example of a reaction involving a concomitant ring expansion and ring extrusion

he ortho methoxycarbonyl substituent constitutes a sole exception in the ring closure reactions of ortho substituted aryl azides, as it provides no rate acceleration to this reaction. Pyrolysis of ''azido-meta-hemipinate'', an aryl azide containing such a substituent, led us to the title compound, a new azepinylidenepyridylacetic ester, whose structure has been established unambiguously by a single crystal X-ray diffraction study. This is the first report of a reaction involving both a ring expansion to an azaheptafulvalene and a ring extrusion to a pyridyl ring residue.

Engineered dimer interface mutants of triosephosphate isomerase: the role of inter-subunit interactions in enzyme function and stability

The role of inter-subunit interactions in maintaining optimal catalytic activity in triosephosphate isomerase (TIM) has been probed, using the Plasmodium falciparum enzyme as a model. Examination of subunit interface contacts in the crystal structures suggests that residue 75 (Thr, conserved) and residue 13 (Cys, variable) make the largest number of inter-subunit contacts. The mutants Cys13Asp (C13D) and Cys13Glu (C13E) have been constructed and display significant reduction in catalytic activity when compared with wild-type (WT) enzyme (similar to 7.4-fold decrease in k(cat) for the C13D and similar to 3.3-fold for the C13E mutants). Analytical gel filtration demonstrates that the C13D mutant dissociates at concentrations < 1.25 mu M, whereas the WT and the C13E enzymes retain the dimeric structure. The order of stability of the mutants in the presence of chemical denaturants, like urea and guanidium chloride, is WT > Cys13Glu > Cys13Asp. Irreversible thermal precipitation temperatures follow the same order as well. Modeling studies establish that the Cys13Asp mutation is likely to cause a significantly greater structural perturbation than Cys13Glu. Analysis of sequence and structural data for TIMs from diverse sources suggests that residues 13 and 82 form a pair of proximal sites, in which a limited number of residue pairs may be accommodated.

Ammonia Channeling in Plasmodium falciparum GMP Synthetase: Investigation by NMR Spectroscopy and Biochemical Assays

GMP synthetase, a class I amidotransferase, catalyzes the last step of the purine biosynthetic pathway, where ammonia from glutamine is incorporated into xanthosine 5'-monophospate to yield guanosine 5'-monnophosphate as the main product. Combined biochemical, structural, and computational studies of glutamine amidotransferases have revealed the existence of physically separate active sites connected by molecular tunnels that efficiently transfer ammonia from the glutaminase site to the synthetase site. Here, we have investigated aspects of ammonia channeling in P. falciparum GMP synthetase using biochemical assays in conjunction with N-15-edited proton NMR spectroscopy. Our results suggest that (1) ammonia released from glutamine is not equilibrated with the external medium (2) saturating concentrations of glutamine do not obliterate the incorporation of external ammonia into GMP, and (3) ammonia in the external medium can access the thioester intermediate when the ATPPase domain is bound to substrates. Further, mutation of Cys-102 to alanine confirmed its identity as the catalytic residue in the glutaminase domain, and ammonia-dependent assays on the mutant indicated glutamine to be a partial uncompetitive inhibitor of the enzyme.

Synthesis and Antioxidant Activity of Peptide-Based Ebselen Analogues

A series of di- and tripeptide-based ebselen analogues has been synthesized. The compounds were characterized by H-1, C-13, and Se-77 NMR spectroscopy and mass spectral techniques. The glutathione peroxidase (GPx)-like antioxidant activity has been studied by using H2O2, tert-butyl hydroperoxide (tBuOOH), and cumene hydroperoxide (Cum-OOH) as substrates, and glutathione (GSH) as a co-substrate. Although all the peptide-based compounds have a selenazole ring similar to that of ebselen, the GPx activity of these compounds highly depends on the nature of the peptide moiety attached to the nitrogen atom of the selenazole ring. It was observed that the introduction of a phenylalanine (Phe) amino acid residue in the N-terminal reduces the activity in all three peroxide systems. On the other hand, the introduction of aliphatic amino acid residues such as valine (Val) significantly enhances the GPx activity of the ebselen analogues. The difference in the catalytic activity of dipeptide-based ebselen derivatives can be ascribed mainly to the change in the reactivity of these compounds toward GSH and peroxide. Although the presence of the Val-Ala-CO2Me moiety facilitates the formation of a catalytically active selenol species, the reaction of ebselen analogues that has a Phe-Ile-CO2Me residue with GSH does not generate the corresponding selenol. To understand the antioxidant activity of the peptide-based ebselen analogues in the absence of GSH, these compounds were studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA) and oxidation of dihydrorhodamine 123. In contrast to the GPx activity, the PN-scavenging activity of the Phe-based peptide analogues was found to be comparable to that of the Val-based compounds. However, the introduction of an additional Phe residue to the ebselen analogue that had a Val-Ala dipeptide significantly reduced the potency of the parent compound in PN-mediated nitration.

The structures of bacteriorhodopsin with different retinal-Schiff base orientations--computer modeling and energy minimization studies

Bacteriorhodopsin has been the subject of intense study in order to understand its photochemical function. The recent atomic model proposed by Henderson and coworkers based on electron cryo-microscopic studies has helped in understanding many of the structural and functional aspects of bacteriorhodopsin. However, the accuracy of the positions of the side chains is not very high since the model is based on low-resolution data. In this study, we have minimized the energy of this structure of bacteriorhodopsin and analyzed various types of interactions such as - intrahelical and interhelical hydrogen bonds and retinal environment. In order to understand the photochemical action, it is necessary to obtain information on the structures adopted at the intermediate states. In this direction, we have generated some intermediate structures taking into account certain experimental data, by computer modeling studies. Various isomers of retinal with 13-cis and/or 15-cis conformations and all possible staggered orientations of Lys-216 side chain were generated. The resultant structures were examined for the distance between Lys-216-schiff base nitrogen and the carboxylate oxygen atoms of Asp-96 - a residue which is known to reprotonate the schiff base at later stages of photocycle. Some of the structures were selected on the basis of suitable retinal orientation and the stability of these structures were tested by energy minimization studies. Further, the minimized structures are analyzed for the hydrogen bond interactions and retinal environment and the results are compared with those of the minimized rest state structure. The importance of functional groups in stabilizing the structure of bacteriorhodopsin and in participating dynamically during the photocycle have been discussed.

Thiol/disulphide exchange reaction: A key regulatory process in biological systems

Cysteine residues in proteins serve many important functions such as stabilizing and maintaining the three-dimensional conformation of many proteins(1), in enzyme catalysis, as a residue undergoing post-translational 2 and in the formation of DNA-binding modification domain of a class of transcriptional activators(3), It is also involved in biological redox coupling(4) and xenobiotic metabolism(5). Disulphide bonds formed by xenobiotic metabolism oxidation of cysteine residues have been used as a probe to study the structure/function relationships of proteins, Introducing novel disulphide bonds in proteins to increase their thermal stability and, therefore, the shelf life is an important goal of protein engineering(6,7), In addition, the thiol group of cysteine residue participates in a reaction termed as thiol/disulphide exchange reaction, the biological significance of this reaction being the theme of this review.

DNA binding properties of some cationic acridine derivatives

Four cationic acridine derivatives have been synthesized. The positively charged amine residue in one of these is connected directly on to the acridine nucleus and in three other acridines, the amines are connected via a 9-CH2 unit to acridine. We have investigated the binding of these acridines with mammalian DNA by absorption titration, UV- and induced-CD spectroscopy and competitive ethidium bromide displacement fluorescence assay. The effects on the DNA duplex denaturation melting temperatures upon binding of each one of these are also examined. The results obtained herein clearly show that the introduction of a -CH2 group in the im mediate vicinity of the interrelation moiety introduces alterations in the DNA binding characteristics of the resulting acridines.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. (C) Munksgaard 1995.

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability

Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez-Mondragon E et al. (2004) Biochemistry43, 3255–3263]. We re-examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7–2.1 Å. Kinetic studies revealed an approximately five-fold drop in kcat for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants. At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (> 40 °C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μm. The mutants also had diminished thermal stability at low concentration, as monitored by far-UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface.