Molecular Mode Of Interaction Research Articles

Studies of the solvent effects on the internal reorganization energy for electron transfer of uracil and its anion with ONIOM

In this paper, the aqueous adiabatic electron affinity (AEA) of Uracil (U) and internal reorganization energy λ i of the self-exchange electron transfer (ET) reaction between Uracil and Uracil anion radical (U − ) in aqueous solution were studied. The effect of the solvation was studied with the recently developed hybrid quantum molecular chemical method, ONIOM. In all calculations, the geometrical optimization for U and U − was performed at B3LYP/6-31++G(d) level. As for the solvent surroundings, the seven water molecules as the first hydration shell were adopted and treated with B3LYP, PM3 and AMBER methods, namely, ONIOM (B3LYP:B3LYP), ONIOM (B3LYP:PM3) and ONIOM (B3LYP:Amber) methods, respectively. The values of AEA for Uracil, predicted by the above three methods, are small positive ones. The geometrical differences between neutral and anion radical molecules of U originate mainly from those of dihedral angles. According to the corresponding dipole moment values, the excess electron in U − should be trapped dominantly by dipole-bound way. The calculated λ i values by ONIOM (B3LYP:B3LYP) and ONIOM (B3LYP:PM3) are close to each other within 0.89%. The λ i value from ONIOM (B3LYP:Amber) is in agreement with the one from SCRF-CPCM very well. Finally, the calculation results of the detailed geometries and molecular interaction mode effect of U and U − and related water molecules in the hydration shell were discussed.

Molecular mode of interaction of plant amine oxidase with the mechanism-based inhibitor 2-butyne-1,4-diamine.

2-Butyne-1,4-diamine (DABI) is a mechanism-based inhibitor of copper-containing plant amine oxidases; the number of turnovers that leads to enzyme inactivation is approximately 20. The product of DABI oxidation is a very reactive aminoallene that reacts with an essential nucleophilic group at the enzyme active site, forming a covalently bound pyrrole and producing an inactive enzyme. The inactivated enzyme shows a new absorption maximum at 295 nm and gives coloured derivatives with p-dimethylaminobenzaldehyde and p-dimethylaminocinnamaldehyde that are spectrally similar to the products of pyrrole treated with the above reagents. Resonance Raman spectra of the p-dimethylaminobenzaldehyde adduct of pyrrole and the inactivated enzyme show very high degree of similarity, supporting the idea that the product of inactivation is indeed a bound pyrrole. The bound pyrrole is formed already in the anaerobic step of the reaction, while the topa semiquinone radical is not affected, as shown by the EPR and stopped-flow absorption measurements. Peptides containing the DABI binding site were obtained by proteolysis of inactivated enzyme, isolated by HPLC and analysed by amino acid sequencing and MS. The crystal structure of the amine oxidase from pea has been determined; inhibition is caused mainly by the highly reactive DABI product, 4-amino-2-butynal, binding to a nucleophilic residue at the entrance to the substrate channel. As other DABI labelled peptides were also found and no free DABI product was detected by MS after complete inhibition of the enzyme, it is likely that the DABI product binds also to other solvent exposed nucleophilic residues on the enzyme surface.

Molecular regulation of phospholamban function and gene expression.

Ca-ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic reticulum. Phospholamban (PLN) functions as an inhibitory cofactor for cardiac Ca-ATPase (SERCA2). To define the molecular mode of interaction between two proteins, interaction sites have been identified. Studies using photoactivated cross-linker and chimeric Ca-ATPase between SERCA2 and nonmuscle Ca-ATPase (SERCA3) indicated that potential binding residues are located just downstream of the active ATPase site (Asp351) of SERCA2. Site-directed mutagenesis study of this region showed that six residues, Lys-Asp-Asp-Lys-Pro-Val402, of SERCA2 are functionally important for the interaction. Further, mutagenesis study of PLN showed that the cytoplasmic region of PLN contains a potential binding site with SERCA2. The unique expression of PLN in cardiac cells has been analyzed by the transcriptional level of its gene using luciferase activity and Gel shift assays. CCAAT-box in the 5'-upstream region was found to be essential for its expression by associating with Y-box binding transcriptional factors.

Insulin signaling in the yeast Saccharomyces cerevisiae. 3. Induction of protein phosphorylation by human insulin.

A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue). Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation. Incubation with [gamma-32P]ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues. In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions. Phosphorylation of both proteins was enhanced by human insulin. These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins. During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins. The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex. Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells.

SR Ca 2+-ATPase/phospholamban incardiomyocyte function

Ca ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic and endoplasmic reticulum (SER). Phospholamban was first identified as a phosphoprotein in cardiac myocytes. Functional properties of phospholamban by steady-state and presteady-state kinetic studies of Ca pump ATPase suggest that phospholamban functions as an inhibitory co-factor for cardiac Ca ATPase (SERCA 2). Protein kinase A-catalyzed phosphorylation of phospholamban results in the dissociation of phospholamban from the Ca ATPase, thus augmenting the ATPase activity. Phospholamban is found as a homo-pentamer, formed from subunits of 6080 Da in size. PKA-catalyzed and CAM kinase- catalyzed phosphorylation residues (Ser 16 and Thr 17) are located in the N-terminal cytoplasmic domain, whereas the C-terminal 22 residues are extremely hydrophobic and are considered to be embedded in the SR membrane. At least three kinds of Ca ATPase have been found. SERCA 1 is expressed in fast-twitch skeletal muscle, while the SERCA 2 gene encodes two alternatively spliced products, SERCA 2a and 2b. SERCA 2a is expressed in cardiac and slow-twitch skeletal muscles; SERCA 2b in smooth muscle and non-muscle tissues. SERCA 3 is expressed in a broad variety of muscle and non-muscle tissues. In vitro expression systems revealed that the functional properties of Ca transport of SERCA 2 are identical to SERCA 1, but not SERCA 3. In particular, the Ca affinity for Ca transport of SERCA 1 or 2 is lowered by co-expression with phospholamban, whereas that of SERCA3 is not. Identification of the interaction sites of phospholamban and SERCA 2 helps definingthe molecular mode of interaction between the two proteins. Photoactivated crosslinking studies indicated that potential binding residues are located just downstream of the active ATPase site (Asp 351) of SERCA 2, but SERCA 3 is devoid of this sequence. If a chimeric Ca ATPase (CH2) is made from SERCA 2 and 3, in which the SERCA 3 region corresponding to the phospholamban-binding sequence of SERCA 2 is introduced into the remainder of the SERCA 2 molecule, then the interaction with phospholamban is lost. These results suggest that this region of SERCA 2 contains amino acids which are involved in the interaction with phospholamban. By site-directed mutagenesis of amino acids of this region, we were able to show that 6 residues, Lys-Asp-Asp-Lys-Pro-Va1402, of SERCA 2 are functionally important for the interaction. When the chimera CH2 was mutated back to SERCA 2 type, mutated CH2 containing these 6 residues of SERCA 2 restored the interaction with phospholamban. Altogether, these 6 residues of SERCA 2 represent the interaction sites for phospholamban. Mutagenesis studies of phospholamban also demonstrated that the hydrophilic, cytoplasmic region of phospholamban contains a potential binding site for SERCA 2. We therefore conclude that the functional interaction between the two proteins occurs in the cytoplasmic region.

Quantitative structure-activity relationships of benzoylphenylurea larvicides: VII. Separation of effects of substituents in the multisubstituted anilide moiety on the larvicidal activity against Chilo suppressalis

The larvicidal activity of benzoylphenylureas with various multiple substitution patterns at the anilide moiety including chlorfluazuron and teflubenzuron was estimated against the rice stem borer, Chilo suppressalis, under conditions in which the oxidative metabolism was eliminated as far as possible by using piperonyl butoxide. The activity was quantitatively analyzed using physicochemical parameters of substituents at each of the anilide aromatic positions. The results indicated that the total hydrophobicity (with an optimum) and electron-withdrawing ability of substituents are favorable to the activity. Small size of substituents was advantageous but the size effect was specific to their positions. The molecular mode of interaction with the possible receptor was suggested so that the compounds may recognize the possible receptor wall from the side of the vicinal pair of ortho and meta positions occupied by smaller substituents, and the receptor site corresponding to the ortho substituent is hydrophilic and constrained.

Stereochemical relationships between two new curare-like toxins and a model of the acetylcholine receptor at the neuromuscular junction

An hypothesis based on molecular modeling is presented concerning the molecular mode of interaction between two newly described curare-like agents — lophotoxin and conotoxin GI — and the acetylcholine receptor at the neuromuscular junction. An experiment critically to test the hypothesis is described.