Studies on the interaction of Nile red with horseradish peroxidase in solution

The interaction of an extrinsic probe (Nile red) with an enzyme (horseradish peroxidase) in solution was investigated using fluorescence techniques. Nile red fluorescence is very environmentally sensitive and the presence of domains of differing polarity within the enzyme was ascertained by the decomposition of the Nile red emission spectrum. Further evidence for the position of the probe inside the enzyme was obtained from a molecular modeling study. A decrease in the emission intensity of the dye during incubation with horseradish peroxidase was explained by the occurrence of resonance energy transfer between the Nile red and the heme group in the enzyme. This was supported by a calculation of the critical transfer distance and a comparison of the fluorescence intensity of the dye in both the holo- and apo-enzyme. These data were then applied to the study of the effect of temperature on the structure of the enzyme, where changes in conformation were elucidated.     

Theoretical study of the surface properties of poly(dimethylsiloxane) and poly(tetrafluoroethylene)

Molecular dynamics (MD) simulations of poly(dimethylsiloxane) (PDMS) and poly(tetrafluoroethylene) (PTFE) were carried out to determine their surface properties and energies. This study helps to gain better insight into the molecular modeling of PDMS and PTFE, in particular how different approaches affect calculations of surface energy. Current experimental and theoretical data were used to further understand the surface properties of PDMS and PTFE as well as to validate and verify results obtained from the combination of density functional theory (DFT) calculations (including periodic boundary conditions) and MD simulations. Detailed analysis of the structure and electronic properties (by calculation of the projected density of states) of the bulk and surface models of PDMS and PTFE was performed. The sensitivity of the surface energy calculation of these two polymers to the chemistry and model preparation was indicated. The balance between the molecular density, weight (which also reflects bond orientation in the surface region), bond flexibility, and intramolecular interactions including bond stretching was revealed to govern the results obtained. In modeling, the structural organization of polymer near a given surface (types and number of end groups and broken bonds due to application of different cut offs of the periodic structure) also significantly affects the final results. Besides the structural differences, certain simulation parameters, such the DFT functionals and simulation boxes utilized, play an important role in determining surface energy. The models used here were shown to be sufficient due to their good agreement with experimental and other theoretical data related to surface properties and surface energies.     

Electronic structure study using density functional theory in organic dendrimers

The electronic and structural properties of pyrrolic ring derivatives were studied using density functional theory (DFT) in terms of their application as organic semiconductor materials in photovoltaic devices. The B3LYP hybrid functional in combination with Pople type 6-31G(d) basis set with a polarization function was used in order to determine the optimized geometries and the electronic properties of the ground state, while transition energies and excited state properties were obtained from time-dependent (TD)-DFT with B3LYP/6-31G(d) calculation. The investigation of pyrrolic derivatives formed by the arrangement of several monomeric units revealed that three-dimensional (3D) conjugated architectures in which the combination of a triphenylamine (TPA) core with π-conjugated rings attached to the core, present the best geometric and electronic characteristics for use as an organic semiconductor material. The highest occupied molecular orbital (HOMO) − lowest unoccupied molecular orbital (LUMO) energy gap was decreased in 3D-structures that extend the absorption spectrum toward longer wavelengths, revealing a feasible intramolecular charge transfer process in these systems. All calculations in this work were performed using the Gaussian 03 W software package.     

Calculation of the relative binding free energy of 2'GMP and 2'AMP to ribonuclease T1 using molecular dynamics/free energy perturbation approaches

We present a calculation of the relative changes in binding free energy between the complex of ribonuclease T1 (RNase Tr) with its inhibitor 2'-guanosine monophosphate (2'GMP) and that of RNase T1-2'-adenosine monophosphate (2'AMP) by means of a thermodynamic perturbation method implemented with molecular dynamics. Using the available crystal structure of the RNase T1-2'GMP complex, the structure of the RNase T1-2'AMP complex was obtained as a final structure of the perturbation calculation. The calculated difference in the free energy of binding (delta delta Gbind) was 2.76 kcal/mol. This compares well with the experimental value of 3.07 kcal/mol. The encouraging agreement in delta delta Gbind suggests that the interactions of inhibitors with the enzyme are reasonably represented. Energy component analyses of the two complexes reveal that the active site of RNase T1 electrostatically stabilizes the binding of 2'GMP more than that of 2'AMP by 44 kcal/mol, while the van der Waals' interactions are similar in the two complexes. The analyses suggest that the mutation from Glu46 to Gln may lead to a preference of RNase T1 for adenine in contrast to the guanine preference of the wild-type enzyme. Although the molecular dynamics equilibration moves the atoms of the RNase T1-2'GMP system about 0.9 A from their X-ray positions and the mutation of the G to A in the active site increases the deviation from the X-ray structure, the mutation of the A back to G reduces the deviation. This and the agreement found for delta delta Gbind suggest that the molecular dynamics/free energy perturbation method will be useful for both energetic and structural analysis of protein-ligand interactions.     

Electronic structure and conformational properties of <Emphasis Type="Italic">1H</Emphasis>-indole-3-acetic acid

The conformational space of 1H-Indole-3-Acetic Acid (IAA) was scanned using molecular dynamics at semiempirical level, and complemented with functional density calculations at B3LYP/6-31G** level, 14 conformers of lowest energy were obtained. Electronic distributions were analyzed at a higher calculation level, thus improving the basis set (B3LYP/6-311++G**). A topological study based on Bader’s theory (AIM: atoms in molecules) and natural bond orbital (NBO) framework performed with the aim to analyze the stability and reactivity of the conformers allowed the understanding of electronic aspects relevant in the study of the antioxidant properties of IAA. Intramolecular hydrogen bonds were found and were characterized as blue-shifting hydrogen bonding interactions. Furthermore, molecular electrostatic potential maps (MEPs) were obtained and analyzed in the light of AIM and NBO results, thus showing subtle but essential features related not only to reactivity but also with intramolecular weak interactions, charge delocalization and structure stabilization.     

Molecular Modelling of Chymotrypsin-Substrate Interactions: Calculation of Enantioselectivity

A method has been developed for the calculation of the enantioselectivity of chymotrypsin catalysed hydrolytic reactions using molecular mechanics and molecular dynamics. Nine different ester substrates, which are hydrolysed by the enzyme over a wide range of reaction rates have been studied. Models of the transition state of the ester hydrolysis were built using computer aided molecular modelling. The energies of the transition state models were calculated by molecular mechanics and molecular dynamics methods. The point charges of the substrates were modelled from known force field parameters and by semiempirical methods. The difference in free energy of activation between the enantiomers of each substrate were compared with experimental values. The calculations approximated the experimental results. The calculated structure of the transition state model of the chymotrypsin catalysed hydrolysis of acetyl-phenylalanine ester was virtually the same as the published crystal structure of a chymotrypsin-trifluoromethyl ketone inhibitor complex (Brady et al., Biochemistry 29: 7600-7607, 1990).     

Solution conformation on bovine growth hormone releasing factor by 1H NMR and molecular modeling.

The structure of bovine growth hormone releasing factor (bGHRF) consisting of 44 amino acids has been studied in CD and 1H nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular modeling. Since bGHRF does not have an ordered structure in water alone, a 30% 2,2,2-trifluoroethanol (TFE) aqueous solvent was used to induce considerable alpha-helical structures, which corresponds to a helical content of approximately 62% as determined by circular dichroism (CD). The secondary structure was obtained from nuclear Overhauser enhancement and 3J(HN alpha) coupling constant in 30% TFE solution. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculation. A set of 267 interproton distances derived from nuclear Overhauser effect correlation spectroscopy (NOESY) experiments and coupling constants were used. From the initial random conformations, 50 distance geometry structures with minimal violations were selected for further refinement. The 14 best structures were obtained after simulated annealing calculation with energy minimization. The structure of bGHRF in 30% TFE solution was characterized by one alpha-helix (residues 8-19), two poorly constrained helices (residues 23-27 and residues 31-34) and a beta I(III)-turn fragment (residues 20-23; phi(i+1) = -53.1 degrees, psi(i+1) = -19.6 degrees, phi(i+2) = -59.9 degrees, psi(i+2) = -20.6 degrees) connected by the segments of less defined structures in N-terminal and omega-shaped flexible C-terminal determined from NOESY cross peaks between helical segment (residues 14-18) and tail fragment (residues 42-44). The obtained structure will play an important role toward the understanding of the structural and functional role of the GHRF.     

The molecular structure of cyclonucleotide hexamer, CoGoCoGoCoGo having a high-anti conformation

Cyclonucleotide hexamer, CoGoCoGoCoGo, was synthesized and crystallized as orthorhombic with space group C222(1), and unit cell dimensions: a = 48.30, b = 41.53, and c = 31.76A. The X-ray diffraction data up to 1.8A resolution were collected, and the crystal structure analysis by molecular replacement technique is now in progress using two energetically adequate left-handed helical models, which are obtained by conformational energy calculation.     

Crystal structure, detonation performance, and thermal stability of a new polynitro cage compound: 2, 4, 6, 8, 10, 12, 13, 14, 15-nonanitro-2, 4, 6, 8, 10, 12, 13, 14, 15-nonaazaheptacyclo [5.5.1.13, 11. 15, 9] pentadecane

A new polynitro cage compound 2, 4, 6, 8, 10, 12, 13, 14, 15-nonanitro-2, 4, 6, 8, 10, 12, 13, 14, 15-nonaazaheptcyclo [5.5.1.1(3,11).1(5,9)] pentadecane (NNNAHP) was designed in the present work. Its molecular structure was optimized at the B3LYP/6-31 G(d,p) level of density functional theory (DFT) and crystal structure was predicted using the Compass and Dreiding force fields and refined by DFT GGA-RPBE method. The obtained crystal structure of NNNAHP belongs to the P-1 space group and the lattice parameters are a = 9.99 ?, b = 10.78 ?, c = 9.99 ?, α = 90.01°, β = 120.01°, γ = 90.00°, and Z = 2, respectively. Based on the optimized crystal structure, the band gap, density of state, thermodynamic properties, infrared spectrum, strain energy, detonation characteristics, and thermal stability were predicted. Calculation results show that NNNAHP has detonation properties close to those of CL-20 and is a high energy density compound with moderate stability.     

Relative binding free energy calculations of inhibitors to two mutants (Glu46----Ala/Gln) of ribonuclease T1 using molecular dynamics/free energy perturbation approaches.

We present free energy perturbation calculations on the complexes of Glu46----Ala46 (E46A) and Glu46----Gln46 (E46Q) mutants of ribonuclease T1 (RNaseT1) with inhibitors 2'-guanosine monophosphate (GMP) and 2'-adenosine monophosphate (AMP) by a thermodynamic perturbation method implemented with molecular dynamics (MD). Using the available crystal structure of the RNaseT1-GMP complex, the structures of E46A-GMP and E46Q-GMP were model built and equilibrated with MD simulations. The structures of E46A-AMP and E46Q-AMP were obtained as a final structure of the GMP----AMP perturbation calculation respectively. The calculated difference in the free energy of binding (delta delta Gbind) was 0.31 kcal/mol for the E46A system and -1.04 kcal/mol for the E46Q system. The resultant free energies are much smaller than the experimental and calculated value of approximately 3 kcal/mol for the native RNaseT1, which suggests that both mutants have greater relative adenine affinities than native RNaseT1. Especially E46Q is calculated to have a larger affinity for adenine than guanine, as we suggested previously from the calculation on the native RNaseT1. Thus, the molecular dynamics/free energy perturbation method may be helpful in protein engineering, directed toward increasing or changing the substrate specificity of enzymes.     

Relationship of the structure of cellulose and certain model compounds to the dynamics of molecular reactions

Using the results of kinetic studies of free radical recombination in cellulose and in some model compounds, some data as to their structure were obtained. Cellulose is characterized by the distribution function of microregions with different energy of molecular interaction. The minimal value of activation energy of macromolecule movement is 37-42 kJ/mol (1.5-2 hydrogen bonds per glucopyranose ring), the maximal one is 63-105 kJ/mol corresponding to the most ordered (crystalline) regions of cellulose. After amorphization of cellulose the warming-up by stages curve points to even distribution by the forces of molecular interaction.     

A structural analytic method on the phase space data of Linac 4 MV photons based on the real world

PurposeIt was given that the characteristics of the fluence distribution and the energy spectrum structure of 4MV photons on the Phase Space (PhSp) plane and a method to analyzing the characteristics.MethodsAfter the PhSp file of 4 MV photons was acquired by the method of Monte Carlo (MC) calculation, the photons recorded by PhSp file were grouped based on the energy bin, and it was analyzed that the spatial distribution and energy spectrum structure of the photons. The photons in each energy group were continually grouped to sub-files according to momentum bin, and the primary and scattered photons could be separated according to the character of the fluence distribution of the photons in the sub-files.ResultsThe energy of 4 MV beam is a continuous spectrum. The energy constituent on a pixel at different distances from the center point is different, and the average energy on the center axis of the field is the highest; The photons with 0–1.0 MeV had 42.6% of all; that with energy more than 3.0 MeV had 11.7%; greater than 4 MeV, just 1.5%. The primary and scattered photons were easy collected according to the distribution characteristics of sub-groups.ConclusionsThe work to acquire and analyze the PhSp file of the 4 MV beam is significant. 4 MV, 6 MV, 8 MV, 10 MV and 15 MV energy beams basically cover the beams of radiotherapy, and a database of the energy beams could be built for the MC related research of other scholars.     

DFT: a dynamic study of the interaction of ethanol and methanol with platinum

The interaction between alcohol molecules and platinum (Pt) was studied using molecular dynamics (MD; Born-Oppenheimer method). Alcohol molecules like ethanol and methanol present a similar molecular structure, with a methyl group (CH₃) at one end and a fragment of hydroxyl (OH) at the other. This fact generates two orientations that are considered in the interaction with Pt. The MD calculation results for these two orientations indicate a preferential orientation due to energy interactions. A plausible reaction mechanism that takes into account the interaction between Pt and alcohol is presented. The charge transference obtained from the Pt–alcohol interaction was also analyzed. The energy for the two orientations was calculated by indicating the preferential orientation. The methyl and hydroxyl groups are involved in heterolytic breakage of hydrogen bonds, joined to a carbon atom in the former and to an oxygen atom in the latter; however, the methyl group reaction seems to be the most important.     

Theoretical analysis on the structure and properties of thiourea dioxide crystal

The equilibrium geometry, electronic structure and optical properties of thiourea S, S-dioxide crystal have been studied using DFT within generalized gradient approximation (GGA) and the local density approximation (LDA), implemented using ultrasoft pseudo-potentials. The optimum bulk geometry is in good agreement with crystallographic data. An analysis of electronic structure, charge and bond order is presented. The energy gap of thiourea dioxide with GGA and LDA calculation is 3.217 or 3.210 eV, respectively, indicating that the compound is an insulator. The calculated absorption spectrum shows a number of absorption peaks, which are believed to be associated with different exciton states, in the fundamental absorption region.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Three-dimensional structure of a glycosphingolipid having a novel carbohydrate linkage, Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta, determined by theoretical calculations

The novel glycosphingolipid, SEGLx (Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta Cer), which was identified by us (Kawakami Y, et al. (1993) J Biochem 114: 677-83), shows a characteristic spectrum on 1H-NMR analysis, in which the anomeric proton resonances of a reducing end galactose and a glucose are split. To elucidate the structural characteristics of SEGLx, we determined its three-dimensional (3D) structure by means of computer simulation, involving such techniques as molecular mechanics (MM2), the semiempirical molecular orbital method (AM1), molecular dynamics (Amber), and computer 3D modelling. With the hypothesis that all OH group(s) of a ceramide participate in intramolecular hydrogen bonds, two kinds of stable conformers, horizontal and right-angled ones, were formed, depending on the ceramide species. The present findings suggest that the chemical species of both the long chain base and fatty acid moieties, mainly the occurrence of OH group(s), affect the chemical shifts of the anomeric proton resonances not only of the reducing terminal galactose but also the penultimate glucose through the formation of intramolecular hydrogen bonds. Computer simulation through theoretical calculation and 3D modelling was shown to be the best means of confirming the results obtained by experimental analysis.     

Solution conformational study of Scyliorhinin I analogues with conformational constraints by two-dimensional NMR and theoretical conformational analysis.

Two analogues of Scyliorhinin I (Scyl), a tachykinin with N-MeLeu in position 8 and a 1,5-disubstituted tetrazole ring between positions 7 and 8, introduced in order to generate local conformational constraints, were synthesized using the solid-phase method. Conformational studies in water and DMSO-d6 were performed on these peptides using a combination of the two-dimensional NMR technique and theoretical conformational analysis. The algorithm of conformational search consisted of the following three stages: (i) extensive global conformational analysis in order to find all low-energy conformations; (ii) calculation of the NOE effects and vicinal coupling constants for each of the low energy conformations; (iii) determining the statistical weights of these conformations by means of a nonlinear least-squares procedure, in order to obtain the best fit of the averaged simulated spectrum to the experimental one. In both solvents the three-dimensional structure of the analogues studied can be interpreted only in terms of an ensemble of multiple conformations. For [MeLeu8]Scyl, the C-terminal 6-10 fragment adopts more rigid structure than the N-terminal one. In the case of the analogue with the tetrazole ring in DMSO-d6 the three-dimensional structure is characterized by two dominant conformers with similar geometry of their backbones. They superimpose especially well (RMSD = 0.28 A) in the 6-9 fragments. All conformers calculated in both solvents superimpose in their C-terminal fragments much better than those of the first analogue. The results obtained indicate that the introduction of the tetrazole ring into the Scyl molecule rigidifies its structure significantly more than that of MeLeu.     

The use of a genetic algorithm search for molecular mechanics (MM3)-based conformational analysis of oligosaccharides

We have implemented a system called glygal that can perform conformational searches on oligosaccharides using several different genetic algorithm (GA) search methods. The searches are performed in the torsion angle conformational space, considering both the primary glycosidic linkages as well as the pendant groups (C-5-C-6 and hydroxyl groups) where energy calculations are performed using the MM3(96) force field. The system includes a graphical user interface for setting calculation parameters and incorporates a 3D molecular viewer. The system was tested using dozens of structures and we present two case studies for two previously investigated O-specific oligosaccharides of the Shigella dysenteriae type 2 and 4. The results obtained using glygal show a significant reduction in the number of structures that need to be sampled in order to find the best conformation, as compared to filtered systematic search.     

Structural requirement for the rapid movement of charged molecules across membranes. Experiments with tetraphenylborate analogues.

Charge-pulse experiments were performed in the presence of structural analogues of tetraphenylborate (TPB) on membranes made of dioleoyl phosphatidylethanolamine and dioleoyl phosphatidylcholine. The analysis of the experimental results using a previously proposed model allowed the calculation of the partition coefficient, beta, and of the translocation rate constant, kappa i. The temperature dependence of the partition coefficients was used to calculate the thermodynamics of the adsorption of the lipophilic ions to the membranes. The analysis of the translocation rate constants obtained at different temperatures yielded detailed information on the free energy of the TPB-analogues within artificial lipid bilayer membranes, and on the activation energy of the translocation rate constants. The adsorption of the different TPB-analogues to the membranes was only slightly affected by their structure, whereas a dramatic influence of the structure on the free energy of the lipophilic ions within the membranes was observed. The free energy of the ions in the membranes decreased from triphenylcyanoborate (TPCB) to tetrakis(3-trifluoromethylphenyl)borate (TTFPB) by more than 31 kJ/mol (7.4 kcal/mol). This could be concluded from the observed increase in the translocation rate constant by almost six orders of magnitude. The change of the free energy in the membrane was used for the estimation of an effective radius of the TPB-analogues with respect to TPB.     

部分镇痛药的分子模拟及其量化计算

采用分子力学和量子化学方法对某些作用于脑啡肽酶的硫乙内酰脲衍生物（YL－2、YL－12、YL－3和YL－11）的化学结构进行了分析模拟和量化计算。优化了各化合物的空间结构,得到了优势构象和优势构象的能量,研究了分子的电荷分布,前线轨道能量等。供助于理论计算可解释脑啡肽酶抑制剂的构效关系,对其活性差异给予较好的分析,可望为合成具有更高药效的无成瘾性镇痛药提供理论指导。