R－藻红蛋白──金（AUC4）和汞（PCMS）重原子衍生物的制备和分析

在获得适合Ｘ射线衍射分析用的Ｒ－藻红蛋白单晶体的基础上,用重原子浸泡法,将晶体浸入含重金属金和汞的０．０５ｍｏｌ／Ｌ磷酸钠－硫酸铵的母液内,经对晶体衍射点强度的分析。找出有明显变化的重原子衍生物。最终得到了与Ｒ－藻红蛋白晶体同晶型的含金和汞的两种重原子衍生物。用面探测仪分别惧了这两种重原子衍生物的衍射数据。通过差值Ｐａｔｔｅｒｓｏｎ图分析,分别确定了重金属金和汞的位置,经对重原子位置参数精化化,给     

植物黄酮二氢杨梅素的提纯及结晶形态研究

显齿葡萄属植物富含黄酮物质。研究对显齿葡萄黄酮提取物二氢杨梅素的提纯及结晶态进行了初步探讨。结果表明:以水作为结晶溶剂进行多次重结晶,可有效去除植物黄酮提取物的杂质,总黄酮含量由原来的86%提高至96.5%。二氢杨梅素在水相中的结晶态多为针状结晶,水相保温结晶呈放射性棒状结晶。在pH4.5的条件下,其溶解度最低可获得较高的回收率。     

Lactoferrin-melanin interaction and its possible implications in melanin polymerization: crystal structure of the complex formed between mare lactoferrin and melanin monomers at 2.7-A resolution

The concentration of melanin determines the intensity of colors of the skin and hair of animals. Melanin pigments are tyrosine-based polymers formed in melanocytes within specialized organelles called melanosomes. In order to understand the mechanism of melanin polymerization, lactoferrin, a basic protein with a pI value of 9.0, has been used to produce melanin. Lactoferrin is a monomeric iron-binding protein with a molecular weight of 80 kDa. The crystals of lactoferrin were soaked in a solution containing dihydroxyphenylalanine (DOPA) and tyrosinase enzyme. These crystals were used for X-ray intensity data collection. The intensity data were collected to 2.7-A resolution to an overall completeness of 91% with an R(sym) of 0.071. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions: a = 85.0 A, b = 99.8 A, c = 103.4 A. The structure was determined by molecular replacement method, using the model of diferric mare lactoferrin, and refined to an R-factor 0.215 (R(free) = 0.287) for all the data to 2.7-A resolution. The final model comprises 5,281 protein atoms from 689 amino acids, 2Fe(3+), 2CO(2-)(3) ions, 2 indole-5,6-quinone molecules (IQ), and 73 water molecules. Two IQ molecules, one in each lobe, bind to lactoferrin. In the C-lobe, the IQ binds in the iron-binding cleft, whereas in the N-lobe, it is located in the side pocket between two alpha-helices, filled with solvent molecules in the native iron-saturated mare lactoferrin. The IQ molecules interact with protein molecule mainly through glutamic acid in both lobes, without significant perturbation to the protein structure. The orientation of N- and C-lobes in the present structure is similar to that observed in the native iron-saturated protein. However, as a result of the binding of IQ molecules, the orientations of the domains N1, N2 and C1, C2 in the two cases differ slightly.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

泛素、泛素链和蛋白质泛素化研究进展

蛋白质泛素化是以泛素单体和泛素链作为信号分子,共价修饰细胞内其他蛋白质的一种翻译后修饰形式。不同蛋白质底物、同一底物的不同氨基酸修饰位点以及同一位点上泛素链连接方式的不同均可导致细胞效应的差异。蛋白质泛素化在真核细胞内广泛存在,除了介导蛋白质的26S蛋白酶体降解途径之外,还广泛参与了基因转录、蛋白质翻译、信号传导、细胞周期控制以及生长发育等几乎所有的生命活动过程。泛素链的形成及其修饰过程的任何失调均可导致生物体内环境的紊乱,从而产生严重的疾病。文中结合实验室研究,综述了泛素的发现历史、基因特点、晶体结构,特别是泛素链的组装过程、结构、功能以及与人类相关疾病关系的新进展,可为这些疾病的治疗靶点和药物靶标的研究提供思路。     

抗冻蛋白结构与抗冻机制

抗冻蛋白(amifreeze proteins,AFPs)是20世纪60年代从极地鱼血淋巴中分离的一种大分子抗冻剂,迄今为止科学工作者已从陆地昆虫、植物、细菌和真菌等各类生物中分离到多种抗冻蛋白,并测得了它们的基因序列及一些晶体结构,近些年的工作主要集中在该类蛋白质抗冻机制的研究上。抗冻蛋白具有广泛的应用前景,它不但可以应用于食物的冷鲜贮存及移植器官的低温保存,还可通过转基因提高经济作物的抗冻能力。     

Prediction of protein loop geometries in solution

The ability to determine the structure of a protein in solution is a critical tool for structural biology, as proteins in their native state are found in aqueous environments. Using a physical chemistry based prediction protocol, we demonstrate the ability to reproduce protein loop geometries in experimentally derived solution structures. Predictions were run on loops drawn from (1)NMR entries in the Protein Databank (PDB), and from (2) the RECOORD database in which NMR entries from the PDB have been standardized and re-refined in explicit solvent. The predicted structures are validated by comparison with experimental distance restraints, a test of structural quality as defined by the WHAT IF structure validation program, root mean square deviation (RMSD) of the predicted loops to the original structural models, and comparison of precision of the original and predicted ensembles. Results show that for the RECOORD ensembles, the predicted loops are consistent with an average of 95%, 91%, and 87% of experimental restraints for the short, medium and long loops respectively. Prediction accuracy is strongly affected by the quality of the original models, with increases in the percentage of experimental restraints violated of 2% for the short loops, and 9% for both the medium and long loops in the PDB derived ensembles. We anticipate the application of our protocol to theoretical modeling of protein structures, such as fold recognition methods; as well as to experimental determination of protein structures, or segments, for which only sparse NMR restraint data is available.     

Proteins of the Bacillus stearothermophilus ribosome: Crystallization of protein L6

Crystals of ribosomal protein L6 from Bacillus stearothermophilus suitable for high resolution structural studies have been obtained. Crystals are hexagonal with space group P6₁22 (or the enantiomorph P6₅22) and cell dimensions a = b = 72.7 Å, c = 124.9 Å. A search for heavy atom derivatives is in progress.     

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.     

Development of a quartz crystal microbalance (QCM) immunosensor for the detection of Listeria monocytogenes

This study presents the development of a QCM immunosensor for the detection of Listeria monocytogenes. A self-assembled monolayer (SAM) of thiosalicylic acid is incorporated for the covalent attachment of antibodies to the gold surface of the piezoelectric crystal. A non-Sauerbrey increase in frequency is observed upon exposure of such a crystal to specific antigen cells. This unexpected response is consistent with the obtained results and is shown to be specific. The sensor can detect L. monocytogenes cells in real time in solution to 1 × 10⁷ cells/ml. The sensor is reusable more than 10 times without detectable loss in activity and shows negligible response to a non-specific pathogen, Bacillus cereus. The lifetime of the thiolated crystal was also investigated.