蛋白质与类药分子的柔性对接

本文利用“禁忌搜索”算法和Ｇｅｈｌｈａａｒ简化能量势函数实现蛋白质与类药分子之间的柔性对接。对包含１００个复合物的检验集进行了计算检验,得到了满意的结果,８９％预测复合物结构的误差小于０．２５ｎｍ。与利用遗传算法进行柔性对接的ＧＯＬＤ程序相比,本方法的成功率高,局限性小,计算时间也短。     

蛋白质-配体柔性对接综述

蛋白质与配体的相互作用在药物发现与筛选、功能食品开发等方面,都具有重要的科学意义。研究蛋白质-配体对接的目的在于通过已知配体和目标蛋白质的三维结构,运用计算手段来预测和评估蛋白质-配体复合物的三维构象,从而更好地理解蛋白质-配体之间的相互作用。随着已解析蛋白质单体结构的数量和计算能力的不断增加,蛋白质-配体对接也越来越现实并可靠。作者对蛋白质-配体柔性对接过程中所涉及的蛋白质的柔性、配体的准备、构象空间的采样方法、打分函数及其后期处理等方面进行了综述。     

预测蛋白质—蛋白质复合物结构的软对接算法

提出了一种有效的软对接算法 ,用于在已知受体和配体三维结构的条件下预测蛋白质 蛋白质复合物的结构。该算法的分子模型基于Janin提出的简化蛋白质模型 ,并在此基础上有所改进。对蛋白质分子表面的柔性氨基酸残基Arg、Lys、Asp、Glu和Met进行了特殊处理 ,通过软化分子表面的方式考虑了它们的侧链柔性。采用双重过滤技术来排除不合理的对接结构 ,此过滤技术是以复合物界面几何互补性和残基成对偏好性为标准提出的。对所得到的构象进行能量优化 ,之后用打分函数对这些结构进行排序 ,挑选出与复合物天然结构接近的构象。该打分函数包括静电、疏水和范德华相互作用能。用此算法对 2 6个复合物进行了结构预测 ,均找到了近天然结构 ,其中有 2 0个复合物的近天然结构排在了前 10位。改进的分子模型可以在一定程度上描述蛋白质表面残基侧链的柔性 ;双重过滤技术使更多的近天然结构保留下来 ,从而提高了算法成功预测的可能性 ;打分函数可以较合理地评价对接结构。总之 ,此种软对接算法能够对蛋白质分子识别的研究提供有益的帮助。     

蛋白质- 核酸对接方法研究进展

分子对接技术作为预测蛋白质-核酸复合物结构的有效方法,为研究在生物学过程中蛋白质-核酸的相互作用提供了重要的工具。本文首先分析了当前蛋白质-核酸对接研究中的主要困难,例如构象变化和核糖磷酸骨架的带电性问题。然后从构象搜索、打分函数、柔性策略三个方面比较和总结了蛋白质-核酸对接中主要的计算方法。最后回顾了蛋白质-核酸对接计算模型的应用,并对未来的工作进行了展望。     

蛋白质复合物结构预测的集成分子对接方法

蛋白质分子间相互作用与识别是当前生命科学研究的热点,分子对接方法是研究这一问题的有效手段．为了推进分子对接方法的发展,欧洲生物信息学中心组织了国际蛋白质复合物结构预测（CAPRI）竞赛．通过参加CAPRI竞赛,逐步摸索出了一套用于蛋白质复合物结构预测的集成蛋白质一蛋白质分子对接方法HoDock,它包括结合位点预测、初始复合物结构采集、精细复合物结构采集、结构成簇和打分排序以及最终复合物结构挑选等主要步骤．本文以最近的CAPRI Target 39为例,具体说明该方法的主要步骤和应用．该方法在CAPRI Target 39竞赛中取得了比较好的结果,预测结构Model 10是所有参赛小组提交的366个结构中仅有的3个正确结构之一,其配体均方根偏差（L_Rmsd）为0．25nm．在对接过程中,首先用理论预测和实验信息相结合的方法来寻找蛋白质结合位点残基,确认CAPRI Target 39A链的A31TRP和A191HIS,B链的B512ARG和B531ARG为可能结合位点残基．同时,用ZDock程序做不依赖结合位点的初步全局刚性对接．然后,根据结合位点信息进行初步局部刚性对接,从全局和局部对接中挑出了11个初始对接复合物结构．进而,用改进的Rosetta Dock程序做精细位置约束对接,并对每组对接中打分排序前200的结构进行成簇聚类．最后,综合分析打分、成簇和结合位点三方面的信息,得到10个蛋白质复合物结构．竞赛结果表明,A191HIS,B512ARG和B531ARG三个结合位点残基预测正确,提交的10个蛋白质复合物结构中有5个复合物受体一配体界面残基预测成功率较高．与其他参赛小组的对接结果比较,表明HoDock方法具有一定优势．这些结果说明我们提出的集成分子对接方法有助于提高蛋白质复合物结构预测的准确率．     

基于点云卷积神经网络的蛋白质柔性预测

目的 蛋白质的柔性运动对生物体各种反应有着重要意义,基于蛋白质的空间结构预测其柔性运动是蛋白质结构-功能关系领域的重要问题.卷积神经网络(convolutional neural network,CNN)在蛋白质结构-功能关系研究中已有成功应用.方法 本研究借鉴计算机视觉研究中PointNet方法的思想,提出了一种蛋白...     

蛋白质中原子与基团的可及性的一种新计算方法及其应用

实现了一种新的蛋白质中原子层次可及面积的计算方法－Ｍｏｎｔｅ－Ｃａｒｌｏ模拟方法。计算了溶菌酶各个原子与功能基团的可及性,并应用于蛋白质结构和功能的研究中。使得对蛋白质性能的预测更准确,提供了一种对蛋白质结构和功能研究的新思路。     

基于SVM的蛋白质二级结构预测

蛋白质结构预测是现代计算生物领域最重要的问题之一,而蛋白质二级结构预测是蛋白质高级结构预测的基础。目前蛋白质二级结构的预测方法较多,其中SVM方法取得了较高的预测精度。重在阐述使用SVM用于蛋白质二级结构预测的步骤,以及与其他方法进行比较时应该注意的事项,为下一步的研究提供参考及启发。     

基于WEB的关键蛋白质预测平台

关键蛋白质是指那些在蛋白质相互作用网络中承担重要作用、移除后会使蛋白质复合物功能丧失并导致生物无法存活的节点。随着蛋白质数据库的不断完善和高通量技术的发展,使得通过计算方法的关键蛋白预测得到广泛应用。针对目前软件多为桌面应用程序、用户难以迅速适应的情况,本文设计并实现了一个基于WEB的关键蛋白质预测平台EssentialProtein Finder（EP Finder）。该平台集成了DC、BC、CC、EC、LAC、SC和NC7种关键蛋白质预测算法,还提供包含SN、SP、PPV、NPV、ACC、F和折刀曲线图在内的7种评估方法。平台对蛋白质网络图、算法运行及评估结果提供了可视化展示。该平台具有良好的扩展性。     

简单的一致性方法预测蛋白质二级结构

蛋白质二级结构预测对于我们了解蛋白质空间结构是至关重要的一步。文章提出了一种简单的二级结构预测方法,该方法采用多数投票法将现有的3种较好的二级结构预测方法的预测结果汇集形成一致性预测结果。从PDB数据库中随机选取近两年新测定结构的57条相似性小于30%的蛋白质,对该方法的预测结果进行测试,其Q3准确率比3种独立的方法提高了1.12—2.29%,相关系数及SOV准确率也有相应的提高。并且各项准确率均比同样采用一致性方法的Jpred二级结构预测程序准确率要高。这种预测方法虽然原理简单,但无须使用额外的参数,计算量小,易于实现,最重要的前提就是必须选用目前准确性比较出色的蛋白质二级结构预测方法。     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: IV. EFFECTS OF 5-FLUOROURACIL, DIHYDROSTREPTOMYCIN, CHLORAMPHENICOL AND ACRIDINE ORANGE ON THE PROCESSES OF GREENING AND DIVISION OF "GLUCOSE-BLEACHED" ALGAL CELLS

1. The "glucose-bleached" cells of Chlorella protothecoides, whichwere obtained by the method described previously, were transferredto a glucose-free medium containing basal mineral nutrientsalone in the dark, and after a certain period of time, the cellsuspension was supplied with urea and light to induce the greeningof cells. At different times before and after the provisionof urea and light, the inhibitors were applied to the cultureto test their effects upon the process of greening.
2. Markedgreening of the glucose-bleached cells occurred aftera lagperiod in the control culture. 5-Fluorouracil inhibitedthecell greening strongly when it was applied at differenttimesbefore the provision of urea and light. When applied aftertheprovision of urea and light, the suppressive effect of 5-fluorouracilgradually decreased with the delay of its application. No inhibitiveeffect was observed when the uracil analogue was added laterthan the 12th hr after the provision of urea and light, thetime around which the chlorophyll formation started in the controlculture. On the other hand, the cell division was much morestrongly affected by 5-fluorouracil. Even when it was appliedat the 18th hr after the provision of urea and light, the celldivision was completely halted, indicating that the greeningand division of the glucose-bleached cells are separate processes.Different mechanisms of action of the uracil analogue towardsthese two processes were suggested.
3. Dihydrostreptomycin showedits strongest suppressive effectwhen added at the beginningof the dark incubation of algalcells in the glucose-free medium,and with the delay of application,its effect was progressivelyreduced, even during the periodof the dark incubation. Thesuppression, however, was stillmarked when it was applied atthe 15th hr.
4. Chloramphenicol was found to inhibit stronglythe chlorophyllformation and protein synthesis, but, to a muchlesser extent,RNA synthesis. Acridine orange suppressed thecell greeningand division at such a low concentration as 1.5µg/ml.
5. Based on these observations it was concludedthat synthesesof nucleic acid and protein are essential processesfor thegreening of the glucose-bleached algal cells. Successiveeventsoccurring in the greening process were discussed.

(Received March 9, 1965; )     

ASSOCIATION OF STRAWBERRY YOGURT SENSORY PROPERTIES WITH PRODUCT COMPOSITION BY PROCRUSTES ANALYSIS

The flavor of eight samples of commercial strawberry yogurt was studied by Free-Choice Profile analysis (FCP). Generalized Procrustes Analysis (GPA) applied to FCP allowed differentiation between samples and highlighted flavor attributes responsible for the observed differences. The relation between sensory and physicochemical datasets was studied by means of GPA. Those samples with higher carbohydrate content were perceived as sweeter, having stronger strawberry flavor, and with more dairy and yogurt flavors. Samples with higher titratable acidity, ash and protein content were perceived as more acidic and higher in intensity of "faulty" or "defective" flavors. Higher moisture content was associated with lower intensity of "dairy" flavors (creamy, dairy, and yogurt) and greater intensity of rancid flavor. It is concluded that, though not often used to this end, GPA is a suitable method to study the relationship of sensory and instrumental measurements.     

蛋白质多态现象的研究及其应用

蛋白质多态现象是生物界比较普遍的现象,有其深刻的生物学意义。蛋白质多态现象的研究是从Smithies(1955)以淀粉凝胶为载体电泳分离血清蛋白、乳蛋白以及蛋清蛋白的成功才得以发展的。     

Rapid quantitation of uncoupling protein in brown adipose tissue mitochondria by a dot immunobinding ("dot blot") procedure: application to the measurement of uncoupling protein in Richardson's ground squirrel, rats, and mice

A dot immunobinding ("dot blot") method for measuring uncoupling protein in brown adipose tissue mitochondria is described. Mitochondrial proteins were solubilized in sodium dodecyl sulfate and applied directly to a nitrocellulose membrane housed in a 96-well microfiltration manifold. Spare binding sites on the nitrocellulose membrane were blocked with bovine serum albumin and then anti-(uncoupling protein) serum was applied. The antigen-antibody complex was detected by the addition of 125I-labelled protein A. Each nitrocellulose "dot" was cut out and its radioactivity was counted. A calibration curve was constructed from purified uncoupling protein standards, taken through the entire procedure. The dot immunobinding method is sensitive (nanogram quantities of uncoupling protein), and in contrast to conventional radioimmunoassay and enzyme-linked immunosorbent assay procedures, it is also rapid and appears to be very robust. The method has been successfully applied to the measurement of uncoupling protein in brown adipose tissue mitochondria of Richardson's ground squirrel, rats, and mice.     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: III. EFFECTS OF MITOMYCIN C ON THE PROCESSES OF GREENING AND DIVISION OF "GLUCOSE-BLEACHED" ALGAL CELLS

The "glucose-bleached" cells of Chlorella protothecoides, whoseplastids were profoundly degenerated containing no trace ofchlorophyll, were obtained by the method previously reported.Transferring the cells to the condition of re-generation ofchloroplasts (greening)—incubation in the light in a glucose-lessand nitrogen-rich medium—the effect of mitomycin C onthe recovery process was investigated. It was found that theantibiotic suppressed completely the cell division without affectingthe re-generation of chloroplasts. De novo formation of RNAand protein which has been observed to occur during the recoveryprocess was not affected by the antibiotic to any significantextent. It thus became clear that the re-generation of chloroplasts,accompanied by the formation of chlorophyll, RNA and protein,occurring under the said condition is not a phenomenon causedby the formation of new "normal" cells from previously degeneratedcells. As was expected, the antibiotic suppressed strongly theDNA synthesis, indicating that the new formation of DNA is nota necessary condition for the re-generation of chloroplastsin "glucose-bleached" algal cells. (Received March 1, 1965; )     

EFFECTS OF GLUCOSE ON THE PROCESS OF CHLOROPLAST DEVELOPMENT IN CHLORELLA PROTOTHECOIDES

By growing Chlorella protothecoides in a medium rich in glucoseand poor in nitrogen source (urea), the entirely chlorophyll-lesscells showing no discernible plastid structure and containingonly little RNA and protein were obtained. These cells, whichwere called "glucosebleached" cells, turned green after a certainlag period, when they were incubated, in the light, in a mediumcontaining the nitrogen source and basal mineral nutrients butwithout glucose. As has been shown in previous studies, thisgreening process involves two consecutive steps : a light-independentphase, in which RNA plays an essential role, and a light-requiringphase, in which the chlorophyll formation and full organizationof chloroplast take place accompanied by the formation of acertain species of protein ("alkali-stable" protein). The wholeprocess of greening was found to be profoundly suppressed byaddition of glucose, the degree of suppression being determinedby the relative concentrations of glucose and the nitrogen source.The primary act of glucose was manifest in the repression ofsyntheses of RNA, and of the species of protein mentioned above,which participate in, or causally related to, the first andsecond phases, respectively, in the greening process. ¹ This paper was read at the Symposium on Extranuclear Self-reproducingSystems held by the Botanical Society of Japan in October, 1964     

生长素极性运输的自动抑制

以豌豆(Pisum sativum L.)和绿豆(Phaseolus radiatus L.)为材料证明了IAA极性运输的自动抑制现象。用改进的“供体-受体技术”证明:受体中的IAAA可抑制~3H-IAA的极性运输,抑制程度随受体中加入IAA浓度的增加而增强;在“Y”型外植体一侧切口施用的IAA也可抑制~3H-IAA在另一侧的极性运输,并导致~3H-IAA在该侧组织中的积累以及代谢或钝化的增强。此外,组织中游离~3H-IAA的比例也随运输时间的延长而不断减小。     

CHANGES OF AMINO ACID COMPOSITION OF CHLORELLA CELLS DURING THEIR LIFE CYCLE

The cells of Chlorella ellipsoidea were grown synchronously,and at different stages of their life cycle, the cells wereanalysed for their contents in amino acids existing in freeforms as well as in the fractions of bulk protein and peptides.Throughout the algal life cycle, the content of bulk protein(per unit dry weight of cells) remained relatively constant,being about 20 to 40 times those of peptides and free aminoacids. The amino acid composition of the protein fraction alsoremained fairly constant, the predominant amino acids beingalanine, glutamic acid, glycine and leucine. The contents inthe bulk peptides increased appreciably during the periods ofgrowth and "ripening" (light period), and decreased markedlyduring the periods of "post-ripening" and cellular division(dark period). Similar modes of change in content were alsoobserved in most of the individual amino acids contained inthe peptide fraction. The most abundant component in the peptidefraction was arginine followed by glutamic acid, glycine andcyst(e)ine. Rather irregular was the mode of change of the levelsof individual free amino acids, although, as a whole, theirbehavior was similar to that of bulk peptides, increasing duringthe light period and decreasing during the dark period. Themost predominant free amino acids were glutamic acid and alaninefollowed by proline. Experimental evidence showed that the processes of formationof free amino acids and peptides are for the most part lightdependent, while the synthesis of protein, which is thoughtto be effected using as building blocks mostly free amino acids—formeddirectly or indirectly from early photosynthates or derivedfrom pre-formed peptides—is essentially a light-independentprocess. Peptides, as a whole, seem to have significance asreservoirs of building blocks for the syntheses in the darkof protein and other nitrogenous cellular substances. The synthesisof protein in the dark takes place not only by consuming thefree amino acids and peptides that have been accumulated duringthe light period, but also by assimilating the exogenous nitrogensource (nitrate). The distribution of individual amino acidsin the three main fractions mentioned above as it changed duringthe course of algal cell cycle was followed in detail, and theresults obtained were discussed in relation to various relevantdata reported by other workers. (Received June 29, 1964; )     

ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-¹⁴C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-¹⁴C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.     

ISOLATION AND CHARACTERIZATION OF THE SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was studied after isolation of a vesicle fraction and of vesicular subfractions by means of differential and density gradient centrifugations. The different fractions were examined electron microscopically by negative and positive staining; their content in protein and phospholipid and their ability to bind Ca⁺⁺ were determined. After homogenization, differential centrifugation yielded a "sarcovesicular fraction" (SVF) which was mainly composed of numerous vesicles of different types mixed with fibrous proteins and mitochondrial fragments. This SVF contained 2% of the protein and 25% of the phospholipid of the initial tissue extract. It had a high Ca⁺⁺ binding activity that was preserved for several days by storage in the presence of oxalate. After centrifugations of the SVF on sucrose density gradients, two vesicular subfractions were obtained which were characterized by different sedimentation rates, isopycnic banding, morphology, and composition in protein and phospholipid. (a) The low-density subfraction (ρ 1.10–1.12) contained a heterogeneous population of membranous structures: thick- and thin-walled vesicles, tubular formations, triads, and plasma membranes. Its content in protein and phospholipid was very low. (b) The high-density subfraction (ρ 1.13–1.17) was a very pure subfraction composed only of thin-walled vesicles. Its content in phospholipid was high and the ratio of phospholipid-phosphorus to protein was about 20. The calcium-binding activity found in the total SVF was recovered only in this latter homogeneous subfraction. The origin of these two subfractions from the SR is discussed.