An evaluation of the performance of an automated procedure for comparative modelling of protein tertiary structure

A 3-D model of a protein can be constructed from its amino acidsequence and the 3-D structures of one or more homologues byannealing three sets of fragments: the structurally conservedregions, structurally variable regions and the side chains.The method encoded in the computer program COMPOSER was assessedby generating 3-D models of eight proteins whose crystal structuresare already known and for which 3-D structures of homologuesare available. In the structurally conserved regions, differencesbetween modelled and X-ray structures are smaller than the differencesbetween the X-ray structures of the modelled protein and thehomologues used to build the model. When several homologuesare used, the contributions of the known structures are weighted,preferably by the square of sequence similarity; this is especiallyimportant when the similarities of the homologues to the modelledstructure differ greatly. The ‘collar’ extensionapproach, in which a similar region of different length in ahomologue is used to extend the framework, can result in a moreaccurate model. If known homologues comprise more than one relatedgroup of proteins and they are both distantly related to theunknown, then alignment of the sequence to be modelled witheach group of homologues facilitates identification of structurallyconserved regions of the unknown and leads to an improved model.Models have root mean square differences (r.m.s.d.s) with thestructures defined by X-ray analysis of between 0.73 and 1.56Å for all C atoms, for seven of the eight models. Forthe model of mucor pepsin, where the closest homologue has 33%sequence identity and 20% of the residues are in structurallyvariable regions, the r.m.s.d. for the framework region is 1.71Å and the r.m.s.d. for all C atoms is 3.47 Â.     

蛋白质在超滤过程中的膜污染和膜清洗

本文选用四种不同的膜清洗剂对牛血清蛋白溶液超滤后污染的三种超滤膜进行研究，结果表明：在蛋白质的等电点取得在不同ＰＨ值下蛋白质对膜污染的最佳清洗效果，清洗效率与蛋白质的所荷电荷有关。采用适宜的清洗过程将会延长膜使用寿命和增强超滤膜性能。     

Vero细胞蛋白过敏原性研究

目的　研究Vero细胞蛋白的过敏原性。方法　取不同剂量Vero细胞宿主细胞蛋白和裂解蛋白致敏豚鼠 ,隔日 1次 ,共 3次 ,每组 3只 ,以牛血清及生理盐水分别为阳性及阴性对照 ,末次致敏后 2 1d攻击 ,并观察攻击后的反应。结果　攻击后 30min ,10 0ng 只以上剂量组均出现过敏反应 ,且反应强度与剂量呈正相关。 2 4h各剂量组过敏反应的恢复情况各不相同。结论　Vero细胞宿主细胞蛋白及裂解蛋白均可以引起过敏反应 ,裂解蛋白的反应强度高于宿主细胞蛋白。     

重组人白细胞介素-10在大肠杆菌中的表达及生物学活性分析

目的在大肠杆菌中表达重组人白细胞介素-10(Interleukin-10,IL-10),并检测其生物学活性。方法将IL-10基因重组到质粒pET11c中,转化BL21(DE),提取质粒,经酶切鉴定和测序分析;在25℃用低浓度的IPTG诱导表达,对包涵体IL-10稀释复性;经ELISA检测其含量,MC/9细胞增殖法检测其生物学活性。结果工程菌IL-10/pET11c/BL21诱导表达的目的蛋白以可溶性和包涵体两种形式存在,Westernblot鉴定证实为IL-10蛋白,两种形式的IL-10均具有一定的生物学活性。结论已成功地在大肠杆菌中表达了IL-10,为进一步纯化和制备IL-10的基因工程药物打下了基础。     

蛋白质化学修饰的研究进展

蛋白质分子具有极其复杂的结构层次，用化学修饰的方法研究蛋白质分子的结构与功能的关系一直是生物化学和分子生物学领域的热点。人们研究出许多小分子化学修饰剂并进行了多种类型的化学修饰。综述了蛋白质化学修饰领域的研究现状与水平，同时强调蛋白质的化学修饰是生化药物研究开发的重要手段之一。     

A molecular dynamics simulation of bacteriophage T4 lysozyme

An analysis of a 400 ps molecular dynamics simulation of the164 amino acid enzyme T4 lysozyme is presented. The simulationwas carried out with all hydrogen atoms modeled explicitly,the inclusion of all 152 crystallographic waters and at a temperatureof 300 K. Temporal analysis of the trajectory versus energy,hydrogen bond stability, r.m.s. deviation from the startingcrystal structure and radius of gyration, demonstrates thatthe simulation was both stable and representative of the averageexperimental structure. Average structural properties were calculatedfrom the enzyme trajectory and compared with the crystal structure.The mean value of the C displacements of the average simulatedstructure from the X-ray structure was 1.1 ± 0.1 Å;differences of the backbone and angles between the averagesimulated structure and the crystal structure were also examined.Thermal-B factors were calculated from the simulation for heavyand backbone atoms and both were in good agreement with experimentalvalues. Relationships between protein secondary structure elementsand internal motions were studied by examining the positionalfluctuations of individual helix, sheet and turn structures.The structural integrity in the secondary structure units waspreserved throughout the simulation; however, the A helix didshow some unusually high atomic fluctuations. The largest backboneatom r.m.s. fluctuations were found in non-secondary structureregions; similar results were observed for r.m.s. fluctuationsof non-secondary structure and angles. In general, the calculatedvalues of r.m.s. fluctuations were quite small for the secondarystructure elements. In contrast, surface loops and turns exhibitedmuch larger values, being able to sample larger regions of conformationalspace. The C difference distance matrix and super-positioninganalyses comparing the X-ray structure with the average dynamicsstructure suggest that a ‘hinge-bending’ motionoccurs between the N- and C-terminal domains.     

Interaction of metal ions with carboxylic and carboxamide groups in protein structures

An analysis of the geometry of metal binding by carboxylic andcarboxamide groups in proteins is presented. Most of the ligandsare from aspartic and glutamic acid side chains. Water moleculesbound to carboxylate anions are known to interact with oxygenlone-pairs. However, metal ions are also found to approach thecarboxylate group along the C - O direction. More metal ionsare found to be along the syn than the anti lone-pair direction.This seems to be the result of the stability of the five-memberedring that is formed by the carboxylate anion hydrogen bondedto a ligand water molecule and the metal ion in the syn position.Ligand residues are usually from the helix, turn or regionswith no regular secondary structure. Because of the steric interactionsassociated with bringing all the ligands around a metal center,a calcium ion can bind only near the ends of a helix; a metal,like zinc, with a low coordination number, can bind anywherein the helix. Based on the analysis of the positions of watermolecules in the metal coordination sphere, the sequence ofthe EF hand (a calcium-binding structure) is discussed.     

人血浆蛋白C的纯化与鉴定

目的　建立人血浆蛋白C的纯化与鉴定方法。方法　利用DEAE SephadexA 5 0代替氯化钡 ,对血浆进行预处理 ,再经DEAE SepharoseFF离子交换和肝素 SepharoseCL 6B亲和层析进行分离纯化 ,用活化的部分凝血活酶时间 (APTT)测定组分活性 ,非还原型SDS PAGE测定其相对分子质量 ,Westernblot鉴定其特异性。结果获得相对分子质量为 6 2 0 0 0的蛋白条带 ,此条带可与人蛋白C单克隆抗体发生特异性反应。结论　已成功地从血浆中分离到蛋白C ,为其规模化生产奠定了基础。     

Lanthanide-binding tags as versatile protein coexpression probes

Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.     

鲁北平原养鸡场周边包气带与地下水砷化合物分布规律

洛克沙胂(3-硝基-4-羟基苯胂酸)被广泛用作饲料添加剂,但大部分以原体形式随粪便排出,进入环境的洛克沙胂在微生物和光照作用下最终转化为毒性较大的As(V)和As(III)等,并在雨水或农灌的淋滤冲积下污染周边土壤、地表水和地下水。以鲁北平原某养鸡场为研究区,对鸡粪、饲料、周边表层土壤、鸡粪堆积和背景包气带垂向剖面以及地下水进行洛克沙胂及其代谢物的检测,结果饲料中洛克沙胂浓度为34mg/kg,鸡粪中HAPA(3-氨基-4-羟基苯胂酸)浓度为11 mg/kg,距离鸡场越近的表层土壤砷含量越高。三个包气带垂向剖面均以As(V)为主,As(III)含量较小。鸡粪堆积处垂向剖面上HAPA、As(V)和As(III)在土壤表层含量最高,浓度随深度增加而呈下降趋势,同时受到土壤岩性的影响,砷化合物主要的吸附层位为0~30cm与200cm以下;背景包气带垂向剖面上各砷化合物含量均明显低于鸡粪堆积处包气带垂向剖面的砷化合物含量;在鸡场内旧井中检测到浓度为165μg/kg的As(V)。结果证实鸡粪堆积导致土壤包气带、周边土壤和地下水砷含量增加,且这种污染很难消除。