Structural forms and transitions for the complex of mercury(II) with poly(dG-dC)

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury(II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N3 or N7 for r less than or equal to 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D2O stabilized the Z structure for poly(dG-dC) complexed with HgCl2.     

A high-resolution structure of a DNA-chromomycin-Co(II) complex determined from pseudocontact shifts in nuclear magnetic resonance

BACKGROUND: The drug chromomycin-A(3) binds to the minor groove of DNA and requires a divalent metal ion for complex formation. (1)H, (31)P and (13)C pseudocontact shifts occurring in the presence of a tightly bound divalent cobalt ion in the complex between d(TTGGCCAA)(2) and chromomycin-A(3) have been used to determine the structure of the complex. The accuracy of the structure was verified by validation with nuclear Overhauser enhancements (NOEs) and J-coupling constants not used in the structure calculation. RESULTS: The final structure was determined to 0.7 A resolution. The structure was compared with a structure obtained in an earlier study using NOEs, in order to assess the accuracy of NOEs in giving global structural information for a DNA complex. Although some basic features of the structures agreed, they differed substantially in the fine structural details and in the DNA axis curvature generated by the drug. The distortion of base-pair planarity that was observed in the NOE structure was not seen in our structure. Differences in drug orientation and hydrogen bonding also occurred. The curvature and elongation of the DNA that was obtained previously was not found to occur in our study. CONCLUSIONS: The use of pseudocontact shifts has enabled us to obtain a high-precision global structure of the chromomycin-DNA complex, which provides an accurate template on which to consider targeting minor groove binding drugs. The effect of such binding is not propagated far along the helix but is restricted to a local kink in the axis that reverts to its original direction within four base pairs.     

Structural Forms and Transitions for the Complex of Mercury(II) with Poly(dG-dC)

Abstract

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury (II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N 3 orN7forr < 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D,0 stabilized the Z structure for poly(dG-dC) complexed with HgCl₂.     

Homology modeling and molecular dynamics simulation of human prothrombin fragment 1.

The crystallographic structure of bovine prothrombin fragment 1 bound with calcium ions was used to construct the corresponding human prothrombin structure (hf1/Ca). The model structure was refined by molecular dynamics to estimate the average solution structure. Accommodation of long-range ionic forces was essential to reach a stable solution structure. The gamma-carboxyglutamic acid (Gla) domain and the kringle domain of hf1/Ca independently equilibrated. Likewise, the hydrogen bond network and the calcium ion coordinations were well preserved. A discussion of the phospholipid binding of the vitamin K-dependent coagulation proteins in the context of the structure and mutational data of the Gla domain is presented.     

Heterogeneous genetic structure in a Fagus crenata population in an old-growth beech forest revealed by microsatellite markers

The within-population genetic structure of Fagus crenata in a 4-ha plot (200 x 200 m) of an old-growth beech forest was analysed using microsatellite markers. To assess the genetic structure, Moran's I spatial autocorrelation coefficient was calculated. Correlograms of Moran's I showed significant positive values less than 0.100 for short-distance classes, indicating weak genetic structure. The genetic structure within the population is created by limited seed dispersal, and is probably weakened by overlapping seed shadow, secondary seed dispersal, extensive pollen flow and the thinning process. Genetic structure was detected in a western subplot of 50 x 200 m with immature soils and almost no dwarf bamboos (Sasa spp.), where small and intermediate-sized individuals were distributed in aggregations with high density because of successful regeneration. By contrast, genetic structure was not found in an eastern subplot of the same size with mature soils and Sasa cover, where successful regeneration was prevented, and the density of the small and intermediate-sized individuals was low. Moreover, genetic structure of individuals in a small-size class (diameter at breast height < 12 cm) was more obvious than in a large-size class (diameter at breast height >/= 12 cm). The apparent genetic structure detected in the 4-ha plot was therefore probably the result of the structure in the western portion of the plot and in small and intermediate-sized individuals that successfully regenerated under the favourable environment. The heterogeneity in genetic structure presumably reflects variation in the density that should be affected by differences in regeneration dynamics associated with heterogeneity in environmental conditions.     

基于空间结构指数的不同森林群落稳定性研究

以长白山杨桦次生林、云冷杉针阔混交林、云冷杉近原始林为研究对象,基于微观经济学中的柯布—道格拉斯生产函数相关理论,以3个常用的空间结构指标(混交度、大小比数、角尺度)为"投入",林分整体空间结构为"产出",构建林分空间结构生产函数,定义了林分空间结构指数(FSSI)并计算了林分空间结构距离(FSSD)。利用这5个指标分析比较不同演替阶段森林群落的空间结构特点,从而评价各演替阶段的稳定性。结果表明:不同演替阶段森林群落的混交度值均不相同,云冷杉近原始林平均混交度最大,云冷杉针阔混交林次之,杨桦次生林最小;大小比数对不同森林群落空间结构影响较小,其值基本为0.49~0.52;3种森林群落水平分布状态为云冷杉针阔混交林和云冷杉近原始林随机分布,杨桦次生林聚集分布;其空间结构指数大小及与理想结构接近程度排序为:云冷杉近原始林云冷杉针阔混交林杨桦次生林。因此,云冷杉近原始林空间结构较好,稳定性最佳。     

Transition of a Compact Intermediate State of Pea Lectin Under the Influence of Different Molecular Weight Polyethylene Glycols

The compact intermediate of the pea lectin found to exist at pH 2.4 was treated with low (PEG-400), medium (PEG-4000) and high (PEG-20,000) molecular weight PEGs. The changes occurring in the secondary structure of the protein were monitored by CD spectropolarimetry in the far-UV range, intrinsic fluorescence was used as a probe to observe the changes in the tertiary structure which is reflected by the changes in the tryptophan environment, further ANS binding studies were made to know the extent of exposure of the hydrophobic patches which is again indicative of the overall changes occurring in the tertiary structure of the protein. It was found that the three PEGs altered the secondary as well as tertiary structure of the pH 2.4 intermediate leading to the formation of three different intermediates. The intermediates were found to have non-native secondary structure as well as non-native tertiary structure. The intermediate formed by the action of PEG-400 was due to the induction of secondary and tertiary structure while the intermediates formed under the influence of PEG-4000 and PEG-20,000 were due to loss in secondary structure and rearrangement in tertiary structure. Also the ANS binding studies showed the absence of any MG or MG-like structures formed in the folding /unfolding pathway induced by PEGs.     

The role of interfacial structured water on the glycoprotein arrangement in liposomes

The effect of perturbing the interfacial water structure in liposomes on the glycoprotein arrangement in the bilayer was investigated. This perturbation was achieved by a series of reagents called structure makers and breakers. The glycoprotein arrangement in the liposomes was determined by fluorescence measurement with 1-anilino-2-naphtalene sulphonate (ANS). A dependence of (n) (number of binding sites for ANS on the glycoprotein molecule) with concentration of structure maker and breaker reagents was observed. The results have been interpreted as a possible new arrangement of membrane-bound glycoprotein, due to the effect of perturbing the interfacial water structure in the liposomes.     

植物炭疽病真菌金属硫蛋白的三级结构预测

根据实验测定的Ⅰ类金属硫蛋白(metallothionein, MT)三级结构的实验数据,给出该类蛋白质的两种特征结构(CXC、CXXC一级结构,半胱氨酸-金属络合簇三级结构)的原子间距离约束条件,然后运用距离几何算法计算出一系列可能的构象.从这些构象中经统计分析筛选出目标函数值显著较小的结构作为所预测蛋白质的三级结构模型.用已知结构的蓝蟹MT对方法进行检验证实其可行性后,对植物炭疽病真菌金属硫蛋白CAP3进行了三级结构预测.     

The Influence of Biotic and Abiotic Factors on Psammic Rotifers in Artificial and Natural Lakes

The effect of sand grain size, porosity and the abundance of algae on the community structure of psammic rotifers was tested in three anthropogenic lakes (Upper Silesia) and compared with three natural ones (West Pomerania). The structure of grain size in the studied beaches of artificial and natural lakes was similar but differences were found in the abundance of algae. The structure of the psammic rotifer community in Upper Silesian lakes was different from natural lakes in West Pomerania. The structure of psammic rotifer communities in anthropogenic reservoirs and lakes (West Pomerania) differed in numbers of species and numbers of psammobiont species, and also diversity index. The most altered structure of rotifer assemblages was observed in the psammon rotifers of the strongly contaminated Dzierżno Duże Lake.     

The ultrastructure of a halobacterial cell in the region of the flagellum outlet

An intracellular structure previously identified in archaeal cells (Halobacterium salinarum) was studied by electron microscopy of single and serial ultrathin sections. This structure was localized under the cytoplasmic membrane near the cellular poles on both sides of the disc-like lamellar structure (DLS), which we found earlier in the motility apparatus of Hb. salinarum. The organization of this structure differs from that of DLS. Morphological characteristics of the new structure coincide with those of bacterial “polar organelle” (PO) described previously. Similar to PO, the new structure exhibited cytochemical staining that reveals the ATPase activity. The bacterial PO is compared with the analogous structure in archaea.     

The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy

Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 Å. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 Å and a single shell thickness of 15 Å. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The “Rossmann canyon” is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.     

小黑麦碳酸酐酶蛋白质三维结构预测

根据已经克隆到的小黑麦碳酸酐酶基因序列,将其概念地翻译成蛋白质的氨基酸序列。利用MEGA4.1、DNAStar5.02、SOPMA、Swiss-Model Workspace和NCBI-VAST等在线软件和服务器对该小黑麦碳酸酐酶(CA)的一级结构、二级结构及三维结构进行了分子结构模型预测,并对其三维结构进行了比对。结果显示,小黑麦碳酸酐酶定位于线粒体内膜和叶绿体类囊体膜上,具有β类碳酸酐酶所特有的保守性基序C-[SA]-D-S-R-[LIVM]-x-[AP];SOPMA预测的二级结构显示,该酶含有α-螺旋(38.61%)、随机卷曲(54.44%)和β-折叠(6.95%)。通过VAST矢量比对工具将小黑麦碳酸酐酶与模板(lekjA)三维结构进行比对,结果显示小黑麦碳酸酐酶与豌豆β碳酸酐酶同型八聚体中的一个单体(lekjA)具有很好的匹配,故推测小黑麦碳酸酐酶全酶也可能是同型八聚体。     

Structural and conformational stability of horseradish peroxidase: effect of temperature and pH

Detailed circular dichroism and fluorescence studies at different pHs have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme c (HRPc). The change in CD in the 222 nm region corresponds to changes in the overall secondary structure of the enzyme, while that in the 400 nm region (Soret region) corresponds to changes in the tertiary structure around the heme in the enzyme. The temperature dependence of the tertiary structure around the heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme. The results suggested that melting of the tertiary structure to a pre-molten globule form takes place at 45 degrees C, which is much lower than the temperature (T(m) = 74 degrees C) at which depletion of heme from the heme cavity takes place. The melting of the tertiary structure was found to be associated with a pK(a) of approximately 5, indicating that this phase possibly involves breaking of the hydrogen-bonding network of the heme pocket, keeping the heme moiety still inside it. The stability of the secondary structure of the enzyme was also found to decrease at pH below 4.5. A 'high temperature' unfolding phase was observed which was, however, independent of pH. The stability of the secondary structure was found to drastically decrease in the presence of DTT (dithiothreitol), indicating that the 'high temperature' form is possibly stabilized due to interhelical disulfide bonds. Depletion of Ca(2+) ions resulted in a marked decrease in the stability of the secondary structure of the enzyme.     

苏云金芽孢杆菌Ⅳ型抗癌晶体蛋白的分子模建

通过同源建模得到了抗癌晶体蛋白Parasporin-4的初始三维结构,利用分子动力学的方法对初始三维结构进行优化,同时分析了模建分子的结构,最后利用Ramachandran plot,结构匹配等方法对模型进行评价。结果显示得到的Parasporin-4结构是具有三个结构域,蛋白模型分子中的键长、键角以及二面角的分布合理,与模版蛋白的主链a碳原子的均方根差RMSD值为0．547742,在合理范围之内,表明Parasporin-4蛋白模型良好。研究结果为抗癌晶体蛋白的抗癌的分子机制及其定向改造提供了结构信息。     

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley & Sons, Inc.     

Adaptability of restrained molecular dynamics for tertiary structure prediction: application to Crotalus atrox venom phospholipase A2

In order to assess the adaptability and/or applicability of the restrained molecular dynamics (RMD) simulation for building a possible tertiary structure of a protein from the X-ray crystal structure of a family reference protein, the tertiary structure prediction of Crotalus atrox venom phospholipase A2 (PLA2) was attempted based on the X-ray crystal structure of bovine pancreatic PLA2. For the formation of secondary and tertiary structures from the fully extended starting structure, the RMD simulation with interatomic distance restraints and torsion angle restraints, which were derived from homologous amino acid sequence regions in the reference protein, was carried out until the molecular system was fully equilibrated. The predicted tertiary structure of C. atrox venom PLA2 was compared with its X-ray crystal structure, and furthermore the utility of this method was discussed by reference to the similar tertiary structure prediction of beta-trypsin from the X-ray crystal structure of an elastase.     

Star structure of antibody-targeted HPMA copolymer-bound doxorubicin: a novel type of polymeric conjugate for targeted drug delivery with potent antitumor effect

The aim of this study was to compare the properties and antitumor potential of a novel type of antibody-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin conjugates with star structure with those of previously described classic antibody-targeted or lectin-targeted HPMA copolymer-bound doxorubicin conjugates. Classic antibody-targeted conjugates were prepared by aminolytic reaction of the multivalent HPMA copolymer containing side-chains ending in 4-nitrophenyl ester (ONp) reactive groups with primary NH(2) groups of the antibodies. The star structure of antibody-targeted conjugates was prepared using semitelechelic HPMA copolymer chains containing only one reactive N-hydroxysuccinimide group at the end of the backbone chain. In both types of conjugates, B1 monoclonal antibody (mAb) was used as a targeting moiety. B1 mAb recognizes the idiotype of surface IgM on BCL1 cells. The star structure of the targeted conjugate had a narrower molecular mass distribution than the classic structure. The peak in the star structure was around 300-350 kDa, while the classic structure conjugate had a peak around 1300 kDa. Doxorubicin was bound to the HPMA copolymer via Gly-Phe(D,L)-Leu-Gly spacer to ensure the controlled intracellular delivery. The release of doxorubicin from polymer conjugates incubated in the presence of cathepsin B was almost twice faster from the star structure of targeted conjugate than from the classic one. The star structure of the targeted conjugate showed a lower binding activity to BCL1 cells in vitro, but the cytostatic activity measured by [(3)H]thymidine incorporation was three times higher than that seen with the classic conjugate. Cytostatic activity of nontargeted and anti-Thy 1.2 mAb (irrelevant mAb) modified HPMA copolymer-bound doxorubicin was more than hundred times lower as compared to the star structure of B1 mAb targeted conjugate. In vivo, both types of conjugates targeted with B1 mAb bound to BCL1 cells in the spleen with approximately the same intensity. The classic structure of the targeted conjugate bound to BCL1 cells in the blood with a slightly higher intensity than the star structure. Both types of targeted conjugates had a much stronger antitumor effect than nontargeted HPMA copolymer-bound doxorubicin and free doxorubicin. The star structure of targeted conjugate had a remarkably higher antitumor effect than the classic structure: a single intravenous dose of 100 microg of doxorubicin given on day 11 completely cured five out of nine experimental animals whereas the classic structure of targeted conjugate given in the same schedule only prolonged the survival of experimental mice to 138% of control mice. These results show that the star structure of antibody-targeted HPMA copolymer-bound doxorubicin is a suitable conjugate for targeted drug delivery with better characterization, higher cytostatic activity in vitro, and stronger antitumor potential in vivo than classic conjugates.     

The structure of the core region of the lipopolysaccharide from Geobacter sulfurreducens

The structure of the core part of the LPS from Geobacter sulfurreducens was analysed. The LPS contained no O-specific polysaccharide (O-side chain) and upon mild hydrolysis gave a core oligosaccharide, which was isolated by gel chromatography. It was studied by chemical methods, NMR and mass spectrometry, and the following structure was proposed. [carbohydrate structure: see text] where Q = 3-O-Me-alpha-L-QuiNAc-(1-->or H (approximately 3:2).