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At least 811 records · Page 45Linked to original sources
PubMed2004

Structural models for the reduced form of vanadate-dependent peroxidases: vanadyl complexes with bidentate chiral Schiff base ligands.

The penta-coordinated vanadyl complexes [VO(ON)(2)] have been obtained by reaction between [VOX(2)] (X = acetylacetonate or chloride) and the Schiff base ligands HON = (R)-sal-am, (R)-Clsal-am and (S)-naph-am, where sal and naph are the salicylidene and naphthalidene moieties, and am derives from phenylethylamine. The three complexes and the ligand (R)-Clsal-am have been structurally characterized. The geometry of the complexes is in-between trigonal-bipyramidal (with the two imine functions in the axis) and square-pyramidal; tau values range from 0.66 to 0.44. Structural and EPR (electron paramagnetic resonance) features are in accord with the coordination environment proposed for the inactive, reduced (V(IV)) form of the bromoperoxidase from the marine brown alga Ascophyllum nodosum.

Crystallography, X-Ray↗
PubMed2000

New structural model for Si/SiO2 interfaces derived from spherosiloxane clusters: implications for Si 2p photoemission spectroscopy

In this Letter, we investigate the Si/SiO(2) interface structure formed by the chemisorption of H8Si8O12 and other spherosiloxane clusters on Si(100). Using transition state calculations, we clearly demonstrate that the clusters do not bond to the Si(100) surface via single vertex attachment as proposed previously, but rather attach via Si-O bond cleavage. This alternative cracked cluster geometry allows us to predict the photoemission features of spherosiloxane clusters on Si(100) without invoking second nearest neighbor effects.

Journal Article↗
PubMed1995

Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.

We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.

Acanthamoeba↗
PubMed1997

Structural model of a cyclic dynorphin A analog bound to dodecylphosphocholine micelles by NMR and restrained molecular dynamics.

The compound c[Cys5,11]dynorphin A-(1-11)-NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the kappa-opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 omega bond contains either a beta-turn from residues Arg6 to Arg9 or an alpha-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 omega bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III beta-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a beta-turn from residues Cys5 and Arg8. In this report we describe the generation of three-dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as kappa-opioid agonists.

Amino Acid Sequence↗
PubMed2002

Structural model of nicotinic acetylcholine receptor isotypes bound to acetylcholine and nicotine.

BACKGROUND: Nicotine is a psychoactive drug presenting a diverse array of biological activities, some positive, such as enhancement of cognitive performances, others negative, such as addiction liability. Ligands that discriminate between the different isotypes of nicotinic acetylcholine receptors (nAChRs) could present improved pharmacology and toxicity profile. RESULTS: Based on the recent crystal structure of a soluble acetylcholine binding protein from snails, we have built atomic models of acetylcholine and nicotine bound to the pocket of four different human nAChR subtypes. The structures of the docked ligands correlate with available biochemical data, and reveal that the determinants for isotype selectivity are relying essentially on four residues, providing diversity of the ligand binding pocket both in terms of Van der Waals boundary, and electrostatic potential. We used our models to screen in silico a large compound database and identify a new ligand candidate that could display subtype selectivity. CONCLUSION: The nAChR-agonist models should be useful for the design of nAChR agonists with diverse specificity profiles.

Acetylcholine↗
PubMed2004

[Fe2(SR)2(mu-CO)(CNMe)6]2+ and analogues: a new class of diiron dithiolates as structural models for the H(ox)Air state of the fe-only hydrogenase.

Low-temperature oxidation of Fe(2)(S(2)C(n)H(2n)(CNMe)(6-x)(CO)x (n = 2, 3; x = 2, 3) affords a family of mixed carbonyl-isocyanides of the type [Fe(2)(S(2)C(n)H(2n)(CO)x(CNMe)(7-x)](2+). The degree of substitution is controlled by the RNC/Fe ratio, as well as the degree of initial substitution at iron, with tricarbonyl derivatives favoring more highly carbonylated products. The structures of the monocarbonyl derivatives [Fe(2)(S(2)C(n)H(2n))(mu-CO)(CNMe)(6)](PF(6))(2) (n = 2,3) established crystallographically and spectroscopically, are quite similar, with Fe---Fe distances of ca. 2.5 A, although the mu-CO is unsymmetrical in the propanedithiolate derivative. Isomeric forms of [Fe(2)(S(2)C(3)H(6))(CO)(CNMe)(6)](PF(6))(2) were characterized where the CO is bridging or terminal, the greatest structural difference being the 0.1 A elongation of the Fe---Fe distance when MeNC (vs CO) is bridging. In the dicarbonyl species, [Fe(2)(S(2)C(2)H(4))(mu-CO)(CO)(CNMe)(5)](PF(6))(2), the terminal CO ligand is situated at one of the basal sites, not trans to the Fe---Fe vector. Oxidation of Fe(2)(S(2)C(2)H(4))(CNMe)(3)(CO)(3) under 1 atm CO gives the deep pink tricarbonyl [Fe(2)(S(2)C(2)H(4))(CO)(3)(CNMe)(4)](PF(6))(2). DFT calculations show that a bridging CO or MeNC establishes a 3-center, 2-electron bond within the two Fe(II) centers, which would otherwise be nonbonding.

Crystallography, X-Ray↗
PubMed2000

Structural modeling of a plant disease resistance gene product domain.

Dominant plant resistance genes are involved in the protection of plants against a wide variety of pathogens. Sequence analysis has revealed a variety of classes, often having domains in common. One commonly found region has come to be known as a putative nucleotide-binding site (NBS) due to the simple presence of sequence motifs. Until now, no experimental evidence has supported this idea. Here we suggest, as an alternative hypothesis, that part of this region is structurally homologous to the receiver domain common to many proteins of His-Asp phosphotransfer pathways. This conclusion is based on sequence analysis, threading experiments, and the construction of a molecular model of one domain that performs well against structure validation tools. The new hypothesis, in contrast to the NBS hypothesis, can explain the devastating effect of a Thr-->Ala mutation in a well-characterized resistance gene product. According to the new hypothesis, regions located N-terminal and C-terminal to the modeled portion, containing highly conserved sequence motifs, could form a separate domain.

Amino Acid Sequence↗
PubMed2006

A model structure for the heterodimer apoA-IMilano-apoA-II supports its peculiar susceptibility to proteolysis.

In this study, we propose a structure for the heterodimer between apolipoprotein A-I(Milano) and apolipoprotein A-II (apoA-I(M)-apoA-II) in a synthetic high-density lipoprotein (HDL) containing L-alpha-palmitoyloleoyl phosphatidylcholine. We applied bioinformatics/computational tools and procedures, such as molecular docking, molecular and essential dynamics, starting from published crystal structures for apolipoprotein A-I and apolipoprotein A-II. Structural and energetic analyses onto the simulated system showed that the molecular dynamics produced a stabilized synthetic HDL. The essential dynamic analysis showed a deviation from the starting belt structure. Our structural results were validated by limited proteolysis experiments on HDL from apoA-I(M) carriers in comparison with control HDL. The high sensitivity of apoA-I(M)-apoA-II to proteases was in agreement with the high root mean-square fluctuation values and the reduction in secondary structure content from molecular dynamics data. Circular dichroism on synthetic HDL containing apoA-I(M)-apoA-II was consistent with the alpha-helix content computed on the proposed model.

Apolipoprotein A-I↗
PubMed1999

NSF N-terminal domain crystal structure: models of NSF function.

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase essential for eukaryotic vesicle fusion. Along with SNAP proteins, it disassembles cis-SNARE complexes upon ATP hydrolysis, preparing SNAREs for trans complex formation. We have determined the crystal structure of the N-terminal domain of NSF (N) to 1.9 A resolution. N contains two subdomains which form a groove that is a likely SNAP interaction site. Unexpectedly, both N subdomains are structurally similar to domains in EF-Tu. Based on this similarity, we propose a model for a large conformational change in NSF that drives SNARE complex disassembly.

Adenosine Triphosphate↗
PubMed1998

Conformational analysis of the interdomain linker of the central homology region of chloroplast initiation factor IF3 supports a structural model of two compact domains connected by a flexible tether.

A peptide corresponding to the interdomain linker of chloroplast IF3 has been synthesized and its structure studied by NMR and CD as a function of temperature and pH. At low temperature and neutral pH the apparent helical content is 25%. pH and ionic strength dependent CD studies demonstrate that sidechain-sidechain interactions stabilize the structure observed at low temperature. The helicity decreases with temperature and above 25 degrees C the peptide is less than 15% helical. These results indicate that the peptide has little intrinsic tendency to form helical structure at physiologically relevant temperatures and strongly suggests that the linker region is flexible in intact chloroplast IF3.

Amino Acid Sequence↗
PubMed2005

Cloning, characterization and structural model of a FatA-type thioesterase from sunflower seeds (Helianthus annuus L.).

The substrate specificity of acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) determines the fatty acids available for the biosynthesis of storage and membrane lipids in seeds. In order to determine the mechanisms involved in the biosynthesis of fatty acids in sunflower seeds (Helianthus annuus L.), we isolated, cloned and sequenced a cDNA clone of acyl-ACP thioesterase from developing sunflower seeds, HaFatA1. Through the heterologous expression of HaFatA1 in Escherichia coli we have purified and characterized this enzyme, showing that sunflower HaFatA1 cDNA encodes a functional thioesterase with preference for monounsaturated acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/K(m)) in catalyzing oleoyl-ACP, both in vivo and in vitro. By comparing this sequence with those obtained from public databases, we constructed a phylogenetic tree that included FatA and FatB thioesterases, as well as related prokaryotic proteins. The phylogenetic relationships support the endosymbiotic theory of the origin of eukaryotic cells and the suggestion that eubacteria from the delta-subdivision were the guest cells in the symbiosis with archaea. These prokaryotic proteins are more homologous to plant FatB, suggesting that the ancient thioesterases were more similar to FatB. Finally, using the available structure prediction methods, a 3D model of plant acyl-ACP thioesterases is proposed that reflects the combined data from direct mutagenesis and chimera studies. In addition, the model was tested by mutating the residues proposed to interact with the ACP protein in the FatA thioesterase by site-directed mutagenesis. The results indicate that this region is involved in the stabilization of the substrate at the active site.

Amino Acid Sequence↗
PubMed1999

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase.

A three-dimensional model of the Streptomyces coelicolor actinorhodin beta-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. (3)H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2002

Quantum chemical calculations on structural models of the catalytic site of chymotrypsin: comparison of calculated results with experimental data from NMR spectroscopy.

Hybrid density functional quantum mechanical calculations were used to study the strength of the hydrogen bond between His(57) N(delta)(1) and Asp(102) O(delta)(1) in chymotrypsin and how it changes along the reaction coordinate. Comparison of experimental shifts with the results of chemical shift calculations on a variety of small molecules, including species containing very strong hydrogen bonds, has validated the overall approach and provided the means for calibrating and correcting the calculated values. Models of the active site of chymotrypsin in its resting state and tetrahedral intermediate state were derived from high-resolution X-ray structures. The distance between His(57) N(delta)(1) and Asp(102) O(delta)(1) in each model was varied between 2.77 A (weak hydrogen bond) and 2.50 A (extremely strong hydrogen bond), and the one-dimensional potential energy surface of the hydrogen-bonded proton (or deuteron/triton) was determined. The zero-point energy, probability distribution, and chemical shift were determined for each distance. Calculated values for NMR chemical shifts, NMR chemical shift differences between (1)H and (3)H, and (2)H/(1)H fractionation factors were compared with published experimental values. Energies provided by the calculations indicated that the hydrogen bond between His(57) N(delta)(1) and Asp(102) O(delta)(1) in the chymotrypsin active site increases in strength by 11 kcal mol(-)(1) in going from the resting state of the enzyme to the tetrahedral intermediate state. This result confirms the hypothesis that the strengthened hydrogen bond plays an important role in lowering the energy of the transition state and, hence, in the catalytic efficiency of the enzyme. Models of the transition state that best fit the experimental data are consistent with a "strong" hydrogen bond between His(57) N(delta)(1) and Asp(102) O(delta)(1) but apparently not a "low-barrier" or "very strong" hydrogen bond.

Binding Sites↗
PubMed2006

A structural model for monastrol inhibition of dimeric kinesin Eg5.

Eg5 or KSP is a homotetrameric Kinesin-5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo-electron microscopy (cryo-EM) of unidirectional shadowed microtubule-Eg5 complexes have been used to identify the stable dimer Eg5-513. The motility assays show that Eg5-513 promotes robust plus-end-directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro. Eg5-513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5-513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5-microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5-513, which reduced microtubule lattice decoration. Comparisons between the X-ray structures of Eg5-ADP and Eg5-ADP-monastrol with rat kinesin-ADP after docking them into cryo-EM 3-D scaffolds revealed structural evidence for the weaker microtubule-Eg5 interaction in the presence of monastrol.

Adenosine Diphosphate↗