谷氨酰胺结合蛋白的分子动力学模拟和自由能计算

谷氨酰胺结合蛋白(Glutamine-binding protein, GlnBp)是大肠杆菌透性酶系统中一个细胞外液底物专一性结合蛋白, 对于细胞外液中谷氨酰胺(Gln)的运输和传递至关重要. 本文运用分子动力学(Molecular dynamics, MD)模拟采样, 考察了GlnBp关键残基与底物Gln之间的相互作用和GlnBp两条铰链的功能差别; 并采用MM-PBSA方法计算了GlnBp与底物Gln的结合自由能. 结果表明: Ph13, Phe50, Thr118和Ile69与底物Gln的范德华相互作用和Arg75, Thr70, Asp157, Gly68, Lys115, Ala67, His156与底物Gln的静电相互作用是结合Gln的主要推动力; 复合物的铰链区85～89柔性大, 对构象开合提供了结构基础; 而铰链区181～185柔性小, 其作用更多是在功能上把底物Gln限制在口袋中; 自由能预测值与实验值吻合. 本研究很好地解释了GlnBp结构与功能的关系, 为进一步了解GlnBp的开合及转运Gln的机制提供了重要的结构信息.     

Structure of the molybdenum site in YedY, a sulfite oxidase homologue from Escherichia coli

YedY from Escherichia coli is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a Mo(IV) model complex. The K-edge energy was indicative of Mo(V) in YedY, in agreement with X- and Q-band electron paramagnetic resonance results, whereas the hSO protein contained Mo(VI). In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of ～2.4 ?), and one (axial) oxo ligand (Mo═O, ～1.7 ?). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (～2.1 ?) or possibly to a shorter Mo-O(-) bond. Yet another oxygen (or nitrogen) at a ～2.6 ? distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a ～2.6 ? distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism.     

Dissociation energetics of the phenol(+)⋯Ar(2) cluster ion: The role of π→H isomerization

The dissociation energetics in the phenol(+)?Ar(2)(2π) cluster ion have been investigated using photoionization efficiency and mass analyzed threshold ionization spectroscopy. The appearance energies for the loss of one and two Ar atoms are determined as ～210 and ～1115?cm(-1), respectively. The difference between the appearance energy for the first Ar ligand in phenol(+)?Ar(2)(2π) and the dissociation energy of the phenol(+)?Ar(π) dimer (535cm(-1)) is explained by the isomerization of one π-bound Ar ligand to the OH binding site (H-bond) upon ionization. The energy difference between phenol(+)?Ar(2)(2π) and phenol(+)?Ar(2)(H/π) could also be estimated to be around 325cm(-1), which corresponds roughly to the difference of the binding energy of a π-bound and H-bound Ar ligands. The binding energy of the H-bound Ar atom in phenol(+)?Ar(2)(H/π) is derived to be ～905cm(-1).     

Switchable Nonlinear Optical Properties of η(5)-Monocyclopentadienylmetal Complexes: A DFT Approach

Density functional theory (DFT) calculations have been carried out to investigate the switching of the second-order nonlinear optical (NLO) properties of η(5)-monocyclopentadienyliron(II) and ruthenium(II) model complexes presenting 5-(3-(thiophen-2-yl)benzo[c]thiophen-1-yl)thiophene-2-carbonitrile as a ligand. The switching properties were induced by redox means. Both oxidation and reduction stimulus have been considered, and calculations have been performed both for the complexes and for the free benzo[c]thiophene derivative ligand in order to elucidate the role played by the organometallic fragment on the second-order NLO properties of these complexes. B3LYP, CAM-B3LYP, and M06 functionals were used for our calculations. The results show some important structural changes upon oxidation/reduction that are accompanied by significant differences on the corresponding second-order NLO properties. TD-DFT calculations show that these differences on the second-order NLO response upon oxidation/reduction are due to a change in the charge transfer pattern, in which the organometallic iron and ruthenium moieties play an important role. The calculated static hyperpolarizabilities were found to be strongly functional dependent. CAM-B3LYP, however, seems to predict more reliable structural and optical data as well as hyperpolarizabilities when compared to experimental data. The use of this functional predicts that the studied complexes can be viewed as acting as redox second-order NLO switches, in particular using oxidation stimulus. The β(tot) value of one-electron oxidized species is at least ～8.3 times (for Ru complex) and ～5.5 times (for Fe complex) as large as that of its nonoxidized counterparts.     

DFT study on the mechanism of water-assisted dihydrogen elimination in group 6 octahedral metal hydride complexes

The reaction of water with octahedral bis-, tris- and tetrakis-(phosphine)tungsten, (phosphine)molybdenum and (phosphine)chromium complexes has been studied using B3LYP/def2-TZVPP level of DFT to elucidate dissociative, associative and hydride migratory insertion mechanisms for hydrogen elimination. In the dissociative mechanism, phosphine dissociation requires 19.3-28.5 kcal mol(-1) of energy. The phosphine-water ligand exchange is endergonic due to poor coordination ability of water to group 6 metals (binding energy 8.8-15.5 kcal mol(-1)). The ligand exchange leads to intermolecular M-HH(2)O dihydrogen interaction and facilitates dihydrogen elimination (G(act) = 6.8-15.5 kcal mol(-1)). In the associative mechanism, a water molecule in the first solvation shell interacts with the M-H bond through a dihydrogen bond (interaction energy 2.7-4.0 kcal mol(-1)) and leads to the elimination of H(2) by forming a hydroxide complex. Compared to the dissociative mechanism, G(act) of associative mechanisms are ～22 kcal mol(-1) higher. In the hydride migratory insertion mechanism, the hydride ligand shifts to the CO ligand (G(act) = 25.4-30.4 kcal mol(-1)) to afford a formyl complex and subsequently the H-H bond coupling occurs between formyl and water ligand (G(act) = 2.8-4.4 kcal mol(-1)). In many cases, the migratory insertion mechanism can simultaneously operate with the dissociative mechanism as a minor pathway, whereas owing to high G(act) value, the associative mechanism can be described as inactive in the reaction. The general argument that dihydrogen elimination is preceded by the formation of a dihydrogen intermediate is not applicable for the systems studied herein as the most favoured dissociative mechanism does not pass through such an intermediate. On the other hand, irrespective of the mechanisms, dihydrogen elimination invariably occurs with the formation of a dihydrogen bonded transition state. Our results also suggest that group 6 octahedral metal hydride complexes are attractive targets for the design of water splitting reactions.     

Ligand coordinate analysis of SC-558 from the active site to the surface of COX-2: a molecular dynamics study

We have performed a ligand coordinate analysis to monitor the movement of the inhibitor SC-558 from the active site of the COX-2 protein to the exterior using molecular dynamics techniques. This study provides an insight into the intermolecular interactions formed by the ligand during this journey. The published crystal structure of COX-2 with SC-558 in the active site (1cx2) was taken, and the ligand was moved incrementally in 13 steps. At each of these points on the path, exhaustive minimization and dynamics calculations were performed. The role of water was found to be important in these computations. An average structure was obtained from 250 conformations at each point and minimized. At each point on the path, the 10 lowest-energy conformations were also selected; a consideration of the average and lowest conformations provides fine details on the consistency of specific and strong interactions, and also on the geometry of the ligand. The movement of the ligand through the protein may be divided into three stages that are distinguished from each other because of energy and geometry discontinuities in both the ligand and the protein. The first of these covers the region between the active site and the point at 5.8 A from it. The second, which covers the distance between 8.2 and 10.0 A and is associated with maximum energetic and structural instability, is of critical importance. The third stage covers the distance between 10.5 A and the exterior and represents a stage of increasing hydration and expulsion of the ligand from the protein. Our results provide a confirmation for the existence of a shallow cavity near the protein surface in which the ligand is bound reversibly. By examining the residues that show maximum mobility, one obtains an idea of the gating mechanism that governs the entry and exit of the protein into or from the deep pocket that contains the active site. We note, however, that the variation of the root-mean-square deviation of all residues begins to increase almost as soon as the ligand leaves the active site, and even before there are any changes in the gate inter-residue distances. This loosening of the protein even before the gate opens might be a part of the enthalpy-entropy balance that accompanies the ligand's passage through the protein. Our results provide an energy profile of the ligand during its entry/exit into/from the protein and can, in principle, enable one to assess the residence time, which in turn may be associated or indirectly correlated with adverse cardiovascular side effects of nonsteroidal anti-inflammatory drugs. We believe that similar analyses for other selected COX-2-specific inhibitors will provide a measure (or prediction) of possible toxicity effects.     

Richardson–Lucy deconvolution of reflection electron energy loss spectra

A procedure for deconvolving the energy spread introduced by the primary beam and the analyzer in a reflection electron energy loss spectrum (REELS) has been developed. The procedure is based on the Richardson–Lucy (RL) algorithm. The approach has been successfully tested on experimental spectra by comparison with spectra with an inherent high‐energy resolution. As a typical result, it was found that the effective energy resolution of spectra with a full width half maximum (FWHM) of the elastic peak of ～1.5 eV in the raw experimental data can be reduced to ～0.7 eV in the deconvoluted spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

HIV-1整合酶与芳香二酮酸类抑制剂相互作用的分子模拟研究

用分子对接方法研究了一系列芳香二酮酸类抑制剂与HIV-1整合酶的识别及相互作用. 结果表明, 抑制剂结合到整合酶Asp64～Leu68, Thr115～Phe121, Gln148～Lys159和Mg2＋所构成的口袋区, 抑制机理与5CITEP相似. 采用分子动力学模拟和MM/PBSA方法计算了芳香二酮酸类抑制剂与整合酶之间的结合自由能, 计算结果与实验值相吻合, 平均绝对偏差为3.6 kJ/mol, 体系范德华相互作用和溶剂化效应的非极性项是利于形成复合物的主要因素. 相关性分析结果表明, 结合自由能值与疏水相互作用有较强的线性相关(R＝0.61), 基于此, 用多元线性回归方法给出了一个能较强预测芳香二酮酸类抑制剂与HIV-1整合酶的结合自由能预测模型, 为后续基于抑制剂结构的抗HIV-1药物分子设计提供指导.     

How to obtain statistically converged MM/GBSA results

The molecular mechanics/generalized Born surface area (MM/GBSA) method has been investigated with the aim of achieving a statistical precision of 1 kJ/mol for the results. We studied the binding of seven biotin analogues to avidin, taking advantage of the fact that the protein is a tetramer with four independent binding sites, which should give the same estimated binding affinities. We show that it is not enough to use a single long simulation (10 ns), because the standard error of such a calculation underestimates the difference between the four binding sites. Instead, it is better to run several independent simulations and average the results. With such an approach, we obtain the same results for the four binding sites, and any desired precision can be obtained by running a proper number of simulations. We discuss how the simulations should be performed to optimize the use of computer time. The correlation time between the MM/GBSA energies is ～5 ps and an equilibration time of 100 ps is needed. For MM/GBSA, we recommend a sampling time of 20–200 ps for each separate simulation, depending on the protein. With 200 ps production time, 5–50 separate simulations are required to reach a statistical precision of 1 kJ/mol (800–8000 energy calculations or 1.5–15 ns total simulation time per ligand) for the seven avidin ligands. This is an order of magnitude more than what is normally used, but such a number of simulations is needed to obtain statistically valid results for the MM/GBSA method. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

Torsion angle preference and energetics of small-molecule ligands bound to proteins

Small organic molecules can assume conformations in the protein-bound state that are significantly different from those in solution. We have analyzed the conformations of 21 common torsion motifs of small molecules extracted from crystal structures of protein-ligand complexes and compared them with their torsion potentials calculated by an ab initio DFT method. We find a good correlation between the potential energy of the torsion motifs and their conformational distribution in the protein-bound state: The most probable conformations of the torsion motifs agree well with the calculated global energy minima, and the lowest torsion-energy state becomes increasingly dominant as the torsion barrier height increases. The torsion motifs can be divided into 3 groups based on torsion barrier heights: high (>4 kcal/mol), medium (2-4 kcal/mol), and low (<2 kcal/mol). The calculated torsion energy profiles are predictive for the most preferred bound conformation for the high and medium barrier groups, the latter group common in druglike molecules. In the high-barrier group of druglike ligands, >95% of conformational torsions occur in the energy region <4 kcal/mol. The conformations of the torsion motifs in the protein-bound state can be modeled by a Boltzmann distribution with a temperature factor much higher than room temperature. This high-temperature factor, derived by fitting the theoretical model to the experimentally observed conformation occurrence of torsions, can be interpreted as the perturbation that proteins inflict on the conformation of the bound ligand. Using this model, it is calculated that the average strain energy of a torsion motif in ligands bound to proteins is approximately 0.6 kcal/mol, a result which can be related to the lower binding efficiency of larger ligands with more rotatable bonds. The above results indicate that torsion potentials play an important role in dictating ligand conformations in both the free and the bound states.     

Can MM/GBSA calculations be sped up by system truncation?

We have studied whether calculations of the binding free energy of small ligands to a protein by the MM/GBSA approach (molecular mechanics combined with generalized Born and surface area solvation) can be sped up by including only a restricted number of atoms close to the ligand. If the protein is truncated before the molecular dynamics (MD) simulations, quite large changes are observed for the calculated binding energies, for example, 4 kJ/mol average difference for a radius of 19 Å for the binding of nine phenol derivatives to ferritin. The results are improved if no atoms are fixed in the simulations, with average and maximum errors of 2 and 3 kJ/mol at 19 Å and 3 and 6 kJ/mol at 7 Å. Similar results are obtained for two additional proteins, p38α MAP kinase and factor Xa. On the other hand, if energies are calculated on snapshots that are truncated after the MD simulation, all residues more than 8.5 Å from the ligand can be omitted without changing the energies by more than 1 kJ/mol on average (maximum error 1.4 kJ/mol). At the molecular mechanics level, the gain in computer time for such an approach is small. However, it shows what size of system should be used if the energies instead are calculated with a more demanding method, for example, quantum‐mechanics. © 2017 Wiley Periodicals, Inc.     

Electrospinning preparation and photoluminescence properties of erbium complex doped composite fibers

In this paper, an Er(III) complex of Er(DBM)3IPD, where DBM=1,3-diphenyl-propane-1,3-dione and IPD=4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-diphenylaniline, is synthesized and doped into poly(vinylpyrrolidone) submicron fibers through electrospinning technique. The crystal structure and morphology are investigated in detail. The composite fibers exhibit smooth and uniform morphology on the substrate, with an average diameter of ～1.4 μm. Photophysical data suggest that DBM ligand sensitizes Er(III) center efficiently and provides an optimal condition for radiative decay, and low temperature can enhance the emission intensity by suppressing the quenching effect. It is found that the photostability of Er(III) complex doped composite fibers is largely improved compared with that of pure complex.     

Translocation of α-helix chains through a nanopore

The translocation of α-helix chains through a nanopore is studied through Langevin dynamics simulations. The α-helix chains exhibit several different characteristics about their average translocation times and the α-helix structures when they transport through the nanopores under the driving forces. First, the relationship between average translocation times τ and the chain length N satisfies the scaling law, τ～N(α), and the scaling exponent α depends on the driving force f for the small forces while it is close to the Flory exponent (ν) in the other force regions. For the chains with given chain lengths, it is observed that the dependence of the average translocation times can be expressed as τ～f(-1/2) for the small forces while can be described as τ～f in the large force regions. Second, for the large driving force, the average number of α-helix structures N(h) decreases first and then increases in the translocation process. The average waiting time of each bead, especially of the first bead, is also dependent on the driving forces. Furthermore, an elasticity spring model is presented to reasonably explain the change of the α-helix number during the translocation and its elasticity can be locally damaged by the large driving forces. Our results demonstrate the unique behaviors of α-helix chains transporting through the pores, which can enrich our insights into and knowledge on biopolymers transporting through membranes.     

Fragmentation energetics of the phenol(+)···Ar(3) cation cluster

The various dissociation thresholds of phenol(+)···Ar(3) complexes for the consecutive loss of all three Ar ligands were measured in a molecular beam using resonant photoionization efficiency and mass analyzed threshold ionization spectroscopy via excitation of the first excited singlet state (S(1)). The adiabatic ionization energy is derived as 68077 ± 15 cm(-1). The analysis of the dissociation thresholds demonstrate that all three Ar ligands in the neutral phenol···Ar(3) tetramer are attached to the aromatic ring via π-bonding, denoted phenol···Ar(3)(3π). The value of the dissociation threshold for the loss of one Ar ligand from phenol(+)···Ar(3)(3π), ～190 cm(-1), is significantly lower than the binding energy measured for the π-bonded Ar ligand in the phenol(+)···Ar(π) dimer, D(0) = 535 ± 3 cm(-1). This difference is rationalized by an ionization-induced π → H isomerization process occurring prior to dissociation, that is, one Ar atom in phenol(+)···Ar(3)(3π) moves to the OH binding site, leading to a structure with one H-bonded and 2 π-bonded ligands, denoted phenol(+)···Ar(3)(H/2π). The dissociation thresholds for the loss of two and three Ar atoms are also reported as 860 and 1730 cm(-1). From these values, the binding energy of the H-bound Ar atom can be estimated as 870 cm(-1).     

Prototype protein assembly as scaffold for time-resolved fluoroimmuno assays

Turnip yellow mosaic virus (TYMV) is an icosahedral plant virus with an average diameter of 28 nm and can be isolated in gram quantities from turnip or Chinese cabbage inexpensively. In this study, it was selected as a prototype bionanoparticle for time-resolved fluoroimmuno assay (TRFIA). Two types of reactive amino acid residues were employed to anchor luminescent terbium complexes and biotin groups based on orthogonal chemical reactions. While terbium complexes were used as luminescent signaling groups, biotin motifs acted as a model ligand for protein binding. The bioconjugation results were confirmed by MS and Western blot analysis. Steady-state and time-resolved luminescence study of the dual-modified viruses demonstrated that the spectroscopic properties of the Tb complex are unperturbed by the labeling procedure. The dual-modified particle was probed by fluorescence resonance energy transfer (FRET) experiments using avidin labeled with an Alexa488 fluorophore, which bound to the biotin on the surface of the particle, as an energy acceptor, and terbium complexes as an energy donor. The emission and excitation spectra of the dual-labeled TYMV particle displayed residual virus fluorescence and Tb luminescence upon ligand-centered excitation. The Tb luminescence lifetime was 1.62 ms and could be effectively fitted with a single-exponential behavior. In the TRFIA, an efficient transfer of 66% was observed, and the calculation using the F?rster radius of 41 A allowed for an estimation of the average donor-acceptor distance of 36 A. Our studies show that the two reactive sites can communicate with each other on the surface of a nanoscale biological assembly. In particular, the ligand-receptor binding (biotin and avidin in this paper) was not interfered with when anchored to the surface of TYMV. Therefore, as a prototype of polyvalent bionanoparticles, TYMV can be used as scaffold for sensor development with TRFIA.     

A model of cooperativity based on restricted binding: kinetic and thermodynamic analysis

Cooperative protein–ligand binding is an essential biochemical process. In this work, we introduce a model that can simulate the emergence of such phenomenon in the binding kinetics. It is based on the inability of the ligand molecules to fully utilize all the available binding sites due to some restriction, realized here in terms of a model parameter, called the restriction parameter. The theory is developed at the level of a single oligomeric protein molecule interacting with a ligand, maintained at a constant concentration, using a chemical master equation. The model provides stepwise binding constants related to the restriction parameter. The relative magnitudes of these constants, when compared to the Hill coefficients measuring cooperativity, give a physical insight in the development of the cooperative behavior and can also act as a reference frame. This can be useful for an alternative theoretical characterization of cooperativity in oligomeric proteins with large number of binding sites and arbitrary binding constants. We establish this point here by taking a tetrameric protein as a case study. A stochastic thermodynamic analysis is also performed, highlighting the energy–entropy contribution to the overall free energy change due to protein–ligand interaction for various cases of restricted binding.     

Indenyl Effect Due to Metal Slippage? Computational Exploration of Rhodium‐Catalyzed Acetylene [2+2+2] Cyclotrimerization

The mechanism of CpRh (Cp=cyclopentadienyl) and IndRh (Ind=indenyl)‐catalyzed acetylene [2+2+2] cyclotrimerization has been revisited aiming at finding an explanation for the better performance of the latter catalyst found experimentally. The hypothesis that an ancillary ligand of the precatalyst remains bonded to the metal center throughout the whole catalytic cycle, based on the experimental evidence that the nature of this ligand can exert some control in cocyclotrimerization of different alkynes, is considered. Strong hapticity variations occur in both the CpRh‐ and IndRh‐catalyzed processes. As the Ind ligand undergoes a more facile slippage than Cp, the energy profile is far smoother in the IndRh‐catalyzed cyclotrimerization. This difference in the energetics of the process translates into an enhanced activity of the IndRh catalyst, in nice agreement with experiment.     

Using the fast fourier transform in binding free energy calculations

According to implicit ligand theory, the standard binding free energy is an exponential average of the binding potential of mean force (BPMF), an exponential average of the interaction energy between the unbound ligand ensemble and a rigid receptor. Here, we use the fast Fourier transform (FFT) to efficiently evaluate BPMFs by calculating interaction energies when rigid ligand configurations from the unbound ensemble are discretely translated across rigid receptor conformations. Results for standard binding free energies between T4 lysozyme and 141 small organic molecules are in good agreement with previous alchemical calculations based on (1) a flexible complex ( for 24 systems) and (2) flexible ligand with multiple rigid receptor configurations ( for 141 systems). While the FFT is routinely used for molecular docking, to our knowledge this is the first time that the algorithm has been used for rigorous binding free energy calculations. © 2017 Wiley Periodicals, Inc.