Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme–Mg-8-oxo-dGMP complex

The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a K_D=52 nM, (ΔG°=−9.8 kcal/mol) which is 6.1 kcal/mol tighter than the binding of dGMP (ΔG°=−3.7 kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable ΔH_binding (−32 kcal/mol) despite an unfavorable −TΔS°_binding (+22 kcal/mol). The solution structure of the MutT–Mg²⁺-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in ¹H–¹⁵N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3 kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1 kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8=O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position −119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3–0.4 kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT–Mg²⁺-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1 kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a single hydrogen bond to its C6=O. The R78K+N119A double mutant weakened the binding of 8-oxo-dGMP (K_I^slope=3.1 mM) by 6.5±0.2 kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0±0.3 kcal/mol). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT–Mg²⁺-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4±0.5 Å between their side chain nitrogens.     

Sequential adsorption of bacteriophage T4 lysozyme stability variants at solid–water interfaces

The sequential adsorption of the wild type T4 lysozyme and one of its structural stability variants was studied, using ellipsometry and ¹²⁵I radioisotope labeling techniques. The mutant lysozyme was produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability. The mutant protein was more resistant to surfactant-mediated elution, and apparently adsorbed at the interfaces with a greater interfacial area/molecule than the wild typeT4 lysozyme. However, the results of each type of experiment suggested that sequential adsorption and exchange of proteins occurred only in the case of the less stable mutant followed by the wild type. This suggests that, in these exchange reactions, properties of the adsorbing protein (e.g. its ability to adsorb when a relatively small amount of unoccupied area is present) were more important than the apparent binding strength of the adsorbed protein molecules.     

Study on the Gas Phase Stability of Heme-binding Pocket in Cytochrome Tb_5 and Its Mutants by Electrospray Mass Spectrometry

Ｃｙｔｏｃｈｒｏｍｅｂ5(Ｃｙｔｂ5)ｉｓｆｏｕｎｄｂｏｔｈａｓａｃｏｍｐｏ ｎｅｎｔｏｆｔｈｅｍｉｃｒｏｓｏｍａｌｍｅｍｂｒａｎｅｓａｎｄａｓａｓｏｌｕｂｌｅｆｏｒｍｉｎｅｒｙｔｈｒｏｃｙｔｅｓ .Ｉｔｐｌａｙｓａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｂｉｏｌｏｇｉｃａｌｓｙｓｔｅｍｓ ,ｉｎｗｈｉｃｈＣｙｔｂ5ｆｕｎｃｔｉｏｎｓａｓａｎｅｌｅｃｔｒｏｎｃａｒｒｉｅｒ,ｐａｒｔｉｃｉｐａｔｉｎｇｉｎａｓｅｒｉｅｓｏｆｅｌｅｃｔｒｏｎ ｔｒａｎｓｆｅｒｐｒｏｃｅｓｓｅｓ ,ｉｎ ｃｌｕｄｉｎｇｒｅｄｕｃｔｉｏｎｏｆ…     

Dynamics of a myoglobin mutant enzyme: 2D IR vibrational echo experiments and simulations

Myoglobin (Mb) double mutant T67R/S92D displays peroxidase enzymatic activity in contrast to the wild type protein. The CO adduct of T67R/S92D shows two CO absorption bands corresponding to the A(1) and A(3) substates. The equilibrium protein dynamics for the two distinct substates of the Mb double mutant are investigated by using two-dimensional infrared (2D IR) vibrational echo spectroscopy and molecular dynamics (MD) simulations. The time-dependent changes in the 2D IR vibrational echo line shapes for both of the substates are analyzed using the center line slope (CLS) method to obtain the frequency-frequency correlation function (FFCF). The results for the double mutant are compared to those from the wild type Mb. The experimentally determined FFCF is compared to the FFCF obtained from molecular dynamics simulations, thereby testing the capacity of a force field to determine the amplitudes and time scales of protein structural fluctuations on fast time scales. The results provide insights into the nature of the energy landscape around the free energy minimum of the folded protein structure.     

Steered Molecular Dynamics for Investigating the Interactions Between Insulin Receptor Tyrosine Kinase (IRK) and Variants of Protein Tyrosine Phosphatase 1B (PTP1B)

The aim of this study is to use steered molecular dynamics to investigate the dissociation process between IRK and PTP1Bs for wild type and five mutants (consisting of p.D181E, p.D181A, p.Q262A, p.D181A-Y46F, and p.D181A-Q262A). The gained results are observed not only the unbinding mechanism of IRK-PTP1B complexes came from pulling force profile, number of hydrogen bonds, and interaction energy between IRK and PTP1Bs but also described PTP1B’s point mutations could variably change its binding affinity towards IRK. Additionally, the binding free energy calculated by Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) is also revealed that electrostatic energy and polar solvation energy mainly made up the binding free energy of PTP1B-IRK complexes.     

Effect of single active-site cleft mutation on product specificity in a thermostable bacterial cellulase

Mutation of a single active-site cleft tyrosyl residue to a glycyl residue significantly changes the mixture of products released from phosphoric acidswollen cellulose (PSC) by EIcd, the catalytic domain of the endoglucanase-I from Acidothermus cellulolyticus. The percentage of glucose in the product stream is almost 40% greater for the Y245G mutant (and for an additional double mutant, Y245G/Q204A) than for the wild type enzyme. Comparisons of results for digestion PSC and of pretreated yellow poplar suggest that the observed shifts in product specificity are connected to the hydrolysis of a more easily digestible fraction of both substrates. A model is presented that relates the changes in product specificity to a mutation-driven shift in indexing of the polymeric substrate along the extended binding-site cleft.     

[Co(pema)(amp)Cl]2^+体系—面式异构体的结构解析及其优势构型

仅合成得到[Co(pema)(amp)Cl]2^+体系配合物的一个面式异构体(pema=N-(2- 吡啶甲基)乙二胺，amp＝2—甲氨基吡啶)．利用二维核磁共振技术与单晶X—ray衍 射法平行解析了该异构体的结构．结果显示结构中存在C—H…π相互作用．用 RHF/LANL2DZ对该体系可能的异构体进行结构、能量优化，可能形成C—H…π相互 作用的异构体具有较好的稳定性．C—H…π相互作用对含吡啶环的[CoN5Cl]^2+系 配合物的异构体的选择性形成及其稳定性具有重要作用．     

细菌视紫红质多重突变体结构变化及其中间态的寿命

采用基因定点突变的方法, 构建了细菌视紫红质(Bacteriorhodopsin, BR)的3种突变体蛋白, 即单突变体BRE194Q、三突变体BRI119T/T121S/A126T和四突变体BRI119T/T121S/A126T/E194Q. 测定了突变体和野生型BR在水溶液和聚乙烯醇(PVA)膜中的紫外-可见吸收光谱和拉曼光谱, 采用显微视频录像技术记录了PVA膜中野生型和3个突变体样品的M态寿命. 与野生型BR相比较, 在水溶液中, 单突变体的可见吸收光谱的最大吸收峰发生了轻微红移, 三突变体和四突变体的最大吸收峰则分别发生了11.0和12.0 nm的明显蓝移. 在PVA膜中, 3个突变体BR的可见吸收光谱的最大吸收峰均发生蓝移, 四突变体BR的最大吸收峰为557 nm, 蓝移达15.0 nm. 四突变体BR在水溶液中的共振拉曼光谱不仅表现有与M态特征相关的1567和1573 cm-1谱带, 还有L态特征带1334 cm-1及N态特征带1200, 1328, 1530和1549 cm-1. 在PVA膜中的样品与在水溶液中的比较, 四突变体共振拉曼光谱的1334和1549 cm-1带消失, 同时1187 cm-1带的强度下降. 显微视频录像技术记录的PVA膜中样品的M态寿命表明, 野生型BR的M态寿命最短, 单突变体的M态寿命小于1.0 s, 三突变体的寿命为3.0 s, 四突变体的寿命为2.0 s.     

Structure of small TiCn clusters: A theoretical study

A theoretical study of the TiC_n (n = 1–8) clusters has been carried out at the B3LYP/6-311+G(d) level. Molecular properties for three different isomers, namely linear, cyclic, and fan species, have been determined. The fan isomers, where the titanium atom is essentially side-bonded to the entire C_n unit, are predicted to be more stable than both linear and cyclic isomers. Only for the largest studied species, TiC₈, the cyclic isomer is located lower in energy. An even–odd parity effect in the incremental binding energies is observed for the three isomers, n-even species being in general more stable for linear and fan isomers, whereas for the cyclic species n-odd clusters are favoured. A topological analysis of the electronic charge density shows that all cyclic isomers correspond to true monocyclic rings, whereas for the fan species a variety of different connectivities has been observed.     

Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis

Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg(2+)-binding domains led in most cases to reductions in both k(cat) and k(cat)/K(m) as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in k(cat) that was offset by a 50-fold decrease in K(m), resulting in a relatively minor 2-fold decrease in catalytic efficiency, k(cat)/K(m). The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4).     

苹果酸酶结合域定点突变对烟酰胺腺嘌呤二核苷酸类似物催化性能的影响

通过对苹果酸酶(ME)辅酶结合域L310、Q401、L404饱和位点突变库与辅酶烟酰胺腺嘌呤二核苷酸(NAD⁺)类似物库的高通量筛选,研究了苹果酸酶结合域位点对NAD⁺及其类似物(B1~B7)催化活性的影响。 结果表明,突变后酶ME-Q401H/L404T对类似物B4的k_cat/K_m是野生型酶的50倍;突变后酶ME-L310M/Q401N对类似物B4的k_cat/K_m是野生型酶的16倍,对类似物B3的k_cat/K_m是野生型酶的5倍,因此通过对结合域定点突变,NAD⁺类似物的催化活性得到提高。     

[Co(2,6-diaminomethylpyridine)(2-methylethylenediamine)Cl][ZnCl_4]体系中几何经式异构体的合成、结构解析和稳定性研究

合成了 [Co(bamp)(cmen)Cl]2＋ (bamp=2,6-二甲胺基吡啶; cmen=1,2-二胺基丙烷 )体系的两个经式异构体。利用一维及二维核磁共振 (2D NMR)技术,结合三元胺中吡啶环的磁屏蔽效应,对两个异构体在溶液中的结构进行了解析,对应于 Dowex 50Wx 2柱色层分离的第一色带的阳离子为 m2(cmen的 2位胺基与 Cl处于邻位 );第二色带的阳离子为 m1。由第一色带配合物制备的单晶晶体结构解析也表明其为 m2[ZnCl4]。用量子化学从头计算方法,在赝势基组 RHF/LANL2DZ的水平上研究了该体系两个经式几何异构体的稳定性,结果表明后者较前者稳定,但差别不大,与在合成条件下异构体平衡分布一致。由计算得到的几何优化键参数与晶体结构分析结果能较好地吻合。     

Ab initio and DFT studies on van der Waals trimers: the OCS.(CO2)2 complexes

Ab initio calculations [MP2, MP4SDTQ, and QCISD(T)] using different basis sets [6-31G(d,p), cc-pVXZ (X = D, T, Q), and aug-cc-pVDZ] and density functional theory [B3LYP/6-31G(d,p)] calculations were carried out to study the OCS.(CO2)2 van der Waals trimer. The DFT has proved inappropriate to the study of this type of systems where the dispersion forces are expected to play a relevant role. Three minima isomers (two noncyclic and one cyclic) were located and characterized. The most stable isomer exhibits a noncyclic barrel-like structure whose bond lengths, angles, rotational constants, and dipole moment agree quite well with the corresponding experimental values of the only structure observed in recent microwave spectroscopic studies. The energetic proximity of the three isomers, with stabilization energies of 1442, 1371, and 1307 cm-1, respectively, at the CBS-MP2/cc-pVXZ (X = D, T, Q) level, strongly suggests that the two unobserved structures should also be detected as in the case of the (CO2)3 trimer where both noncyclic and cyclic isomers have been reported to exist. The many-body symmetry-adapted perturbation theory is employed to analyze the nature of the interactions leading to the formation of the different structures. The three-body contributions are small and stabilizing for the two most stable structures and almost negligible for the cyclic isomer.     

Role of Enterobactin and Intracellular Iron in Cell Lethality During Near-UV Irradiation in Escherichia coli

Abstract— In Escherichia coli, fur mutants that constitutively express their native iron chelating agent, enterobactin, are significantly more sensitive to near-UV radiation (NUV) than wild type. An entA mutant, which is incapable of synthesizing enterobactin, is equal to wild type in resistance to NUV irradiation. However, the addition of Fe⁺³ enterobactin but not Al⁺¹ enterobactin to entA cell suspensions just prior to irradiation results in an increased sensitivity to NUV irradiation. A fes mutant, which is unable to reduce and release iron from enterobactin, is significantly more sensitive to NUV irradiation than wild type. The addition of nontoxic levels of H₂O₂ (5 μ M ) just prior to irradiation significantly increases sensitivity of both fur and fes mutants. These results suggest that one mechanism by which NUV irradiation leads to cell lethality is by creating a transient iron overload, producing very favorable conditions for the production of highly deleterious free radicals through a variety of mechanisms that lead to oxidative stress and DNA damage including lethal and mutagenic lesions. These results are consistent with the hypothesis that enterobactin is an endogenous chromophore for NUV and contributes to cell lethality via the destruction of its ligand, releasing Fe⁺² into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals and other toxic oxygen species via the Haber-Weiss reaction.     

Asymmetric oxidation catalyzed by myoglobin mutants

The sperm whale myoglobin active site mutants (L29H/H64L and F43H/H64L Mb) have been shown to catalyze the asymmetric oxidation of sulfides and olefins. Thioanisole, ethyl phenyl sulfide, and cis-β-methylstyrene are oxidized by L29H/H64L Mb with more than 95% enantiomeric excess (% ee). On the other hand, the F43H/H64L mutant transforms trans-β-methylstyrene into the trans-epoxide with 96% ee. The dominant sulfoxide product in the incubation of alkyl phenyl thioethers is the R isomer; however, the mutants afford dominantly the S isomer of aromatic bicyclic sulfoxides. The results help us to rationalize the difference in the preferred stereochemistry of the Mb mutant-catalyzed reactions. Furthermore, the Mb mutants exhibit an improvement in the oxidation rate up to 300-fold with respect to wild type.     

Strain effects in electron spin resonance spectroscopy of quintet 2,6-bis(4'-nitrenophenyl)-4-phenylpyridine

Photolysis of 2,6-bis(4'-azidophenyl)-4-phenylpyridine in 2-methyltetrahydrofuran (2MTHF) glass at 7 K leads to quintet 2,6-bis(4'-nitrenophenyl)-4-phenylpyridine as a mixture of rotational isomers. The electron spin resonance (ESR) spectrum of this mixture of rotamers shows a considerable broadening of many transitions in the range of 0-5000 G and cannot be reproduced by computer simulations solely based on the tuning of the spin Hamiltonian parameters g, D(Q), and E(Q) alone or on predictions of DFT calculations. The best modeling of the experimental ESR spectrum is obtained only when the large line-broadening parameter of Γ(E(Q)) = 1200 MHz along with the spin Hamiltonian g = 2.003, D(Q) = 0.154 cm(-1), and E(Q) = 0.050 cm(-1) is used in the spectral simulations. The most accurate theoretical estimations of the magnetic parameters of the dinitrene in a 2MTHF glass are obtained from the B3LYP/6-311+G(d,p)+PBE/DZ/COSMO calculations of the spin-spin coupling parameters D(SS) and E(SS). Such calculations overestimate the E(Q) and D(Q) values of the dinitrene just by 1% and 10%, respectively, demonstrating that contributions of the spin-orbit coupling parameters D(SOC) and E(SOC) to the total D(Q) and E(Q) values are negligibly small. The research shows that ESR studies of polynuclear high-spin nitrenes, obtained by photolysis of rotational isomers of the starting azides, can only be successful if large E(Q) strain effects are taken into account in the spectral simulations.     

A unique spherical molecular host with D2d symmetry. A novel intramolecular kinetic equilibrium in metal ion complexation between two crown ethers

[formula: see text] A spherical host with D2d symmetry consisting of a tetrathia[3.3.3.3]paracyclophane and two 18-crown-6 moieties was synthesized. Its crystal structure shows a central cavity with a diameter of 1.96 A and a depth of 6.75 A. A Na+ ion could rest in the cavity center but prefers core binding to external binding in one of the crown units. An intramolecular kinetic equilibrium was reached with the Na+ ion switching between the two crown units with an energy barrier of 14.1 +/- 3 kcal/mol.     

Li3-O-Li3 molecule: a metal-nonmetal-metal sandwichlike compound with a distending electron cloud

The D3d and D2d isomers of the Li3-O-Li3 molecule are metal-nonmetal-metal sandwichlike structures that contain two Li3 superalkali atoms. Their geometries and the real frequencies are obtained at the CCSD(T)/aug-cc-pVDZ level. They are different from the traditional types of the nonmetal-metal-nonmetal sandwich compounds. The natural bond orbital calculation and the topological property nabla2rho(r) calculation indicate that they are typical ionic compounds. In two isomers, the O2- anion is sandwiched in between two Li3+ cation rings. However, the different orientations of two Li3+ planes give the D3d isomer its own special characteristics. Under the action of the O2- anion in the center, the valence electrons of the D3d isomer are pushed out from two Li3+ triangle rings. This special interaction causes three phenomena. First, the valence electron clouds are distended. Second, the vertical ionization energy of the D3d isomer is considerably low, 4.39 eV, so that it may also be viewed as a superalkali atom. Third, we find that the D3d isomer owns the out-of-plane aromaticity and the largest negative nucleus-independent chemical shift value (-10.8 ppm) exists at 2.5 A above the center of the Li3+ ring, not at the center of the Li3+ ring like the isolated aromatic Li3+ cation.     

Site-specific addition of D(2)O to the (H(2)O)(6)(-) "hydrated electron" cluster: isomer interconversion and substitution at the double H-bond acceptor (AA) electron-binding site

We report the results of an experimental study designed to establish whether, once formed, one of the isomer classes of the hydrated electron clusters, (H(2)O)(n)(-), can interconvert with others when a water molecule is added by condensation. This is accomplished in an Ar-cluster mediated approach where a single intact D(2)O molecule is collisionally incorporated into argon-solvated water hexamer anions, creating the isotopically labeled D(2)O.(H(2)O)(6)(-).Ar(n) heptamer anion. Photoelectron and infrared predissociation spectroscopies are employed both to characterize the isomers generated in the condensation event and to track the position that the D(2)O label adopts within these isomeric structures. Despite the fact that the water hexamer anion precursor clusters initially exist in the isomer I form, incorporation of D(2)O produces mostly isomers I' and II in the labeled heptamer, which bind the electron more (I') or less (II) strongly than does the isomer I class. Isomers I and I' are known to feature electron binding primarily onto a single water molecule that resides in an AA (A = H-bond acceptor) site in the network. Surprisingly, the D(2)O molecule can displace this special electron-binding H(2)O molecule such that the anionic cluster retains the high binding arrangement. In the more weakly binding isomer II clusters, the D(2)O molecule fractionates preferentially to sites that give rise to the vibrational signature of isomer II.