shininglake的快乐生活

第一次参加合唱团俱乐部活动

第一次参加活动就碰上暴雨，原本想等小一点再上路，没想到半路上却碰到最猛的一阵。

老婆说这种鬼天气一定没几个人去，没想到竟然到了二十来个！

听了高手的表演发现原来自己还很弱啊！

出线了！

居然0：0逼平号称有多位校队明星的工程院队，居然能抽到好签将他们淘汰！太让人兴奋了。

可怜我的伤腿在场上只坚持了20分钟。一定要去做理疗了，争取下一场能坚持全场！

看到很多好文章

开始学习EMBOSS

先学常用的，然后一个一个来。

在91.118看得头晕，结果在网上找到另一个提供EMBOSS的网站：http://ngfnblast.gbf.de/uk/emboss.html

发现简直是太好用了，界面比咱院的方便多了，以后就用这个了！

老婆来电话了

用“网文快捕”破解不能复制文字的网站

生物通新闻中心（http://www.ebiotrade.com/newsf/readnews.asp?recordno=L2004510115556）的文章不能复制，因为文字不能复制，另存后用FrontPage也不行，后来发现“网文快捕”才行，而且不是用普通的方法，因为右键也不起作用，需要打开“网文快捕”后，右键点“网文快捕”的小方块图标，选择“添加任务”，然后贴上该文的网址，这样文章就被保存，这时文字就可以选择了，复制到WORD就OK了！

更简单一点的办法是在退后一页，在该链接处点右键用网文快捕保存该链接。

文献阅读2——从结构预测功能

Current Opinion in Biotechnology 2000,

Biological function made crystal clear — annotation of hypothetical proteins via structural genomics

该课题组通过解析脑膜炎菌（Haemophilus influenzae）的50个假想蛋白的结构来推断其功能。在基因组序列中发现一些ORF在不同物种中保守存在，然而找不到与其相关的已知结构和功能的蛋白，因此视为假想蛋白（hypothetical protein），对应的基因被称为‘orphan’ genes。

从1743个ORF中选出65个靶目标。选择的标准是：1. 功能未知。2. 可溶（通过TopPhred预测少于3个跨膜区）；然后排除可能预测错误的ORF，标准是在至少3种微生物中都存在；最后一步是用BLAST/BEAUTY、FASTA搜索PROSITE、Swiss Prot、GeneQuiz。每个月要对PDB搜索一次，看看有没有新进展。

 从克隆到结构解析的过程：

克隆与表达是常规的，在大肠杆菌中表达，每个ORF都用3种载体，分别用Native和His－Tag的形式，发现25％不能或少量表达。

结晶方面是用机器人，先快速筛选，如不能找到合适条件再用更多条件试。pH2-10,温度6－35。找到合适的条件再优化。得到晶体后先进行初步衍射试验，加防冻剂等，然后用同步辐射加速器进行X－ray晶体衍射。采用多波长反常衍射方法（MAD）。

NMR的优点是在蛋白在可溶状态下确定结构，然而它只能观察小蛋白（<30kD）,并且要高度可溶（毫摩尔浓度）。

 下面是如何从结构推断功能：蛋白质的结构提供了直接和间接的线索来推断其功能，采用以下四个步骤：

Case 1: the protein has a fold that has been seen before 该蛋白具有已知的折叠（fold）如该折叠与一种或几种生物学功能相联系，则可以对该蛋白的功能做一评估。

Case 2: the protein is an enzyme 该蛋白是一种酶许多酶有着相似的催化机制，如Ser–His–Asp这样的three-dimensional catalytic motifs, 可以搜索新结构中是否有这样的motif.

Case 3: the protein binds one or more small-molecule ligands 该蛋白与一个或多个小分子配体结合。结合位点几乎总是蛋白表面最大的凹陷处，可以通过计算机自动识别。这也是以结构为基础的药物设计的靶标。

Case 4: the protein interacts with other macromolecules 该蛋白与其他大分子相互作用根据相互作用的性质，有5种方法来确定该蛋白是否与其他大分子相互作用。 First, in cases where electrostatics plays a major role in binding, such as when a protein associates with RNA or DNA, mapping of the surface potential can be an effective technique . 表面势能。 Second, sites of tight association with other proteins may be identified by analyses of surface composition. Third, where a large family of sequences are available, mapping the extent of conservation of surface residues provides a means of identifying interaction sites. Fourth, three new genome-scale non-structure-based methods hold promise for providing clues to identifying interacting proteins . Hypotheses generated by these methods may be tested structurally by protein–protein docking methods that search for specific binding sites . Fifth, it has also been suggested that interaction sites can also be identified by analyses of the correlation of sequence changes between pairs of proteins across many species. These cases can also be clarified using protein–protein docking methods.

通过各种方法预测其功能后再用相应的实验来证实。

有时配体会与蛋白共纯化，共结晶，这会提供某种线索。如某蛋白结合有ATP，则该蛋白可能是ATP酶或是由ATP调节的分子开关。　

膜蛋白研究新技术

在三藩市举行的Materials Research Society的一个会议上，来自University of Illinois, Urbana-Champaign的生化学家Stephen Sligar报告说他们的研究小组他们制备出一种脂质体的“超微小碟nanodiscs”并成功将特定的膜结合蛋白嵌入其中，并使之继续工作。这种nanodiscs和天然的细胞膜的组成一样，是由两层磷脂层组成的，每个磷脂分子都有活跃的亲水的头部基团和长长的疏水尾部。为了保持这个nanodiscs为扁平的形状，研究人员给这个nanodiscs外面加上一圈蛋白质，就像紫菜寿司中包着饭团的紫菜卷。

为了证实嵌入这个nanodiscs中的受体可以继续工作，研究人员采用一个已知的蛋白——β2 肾上腺素受体（β2 adrenergic receptor ，简称β2AR，是心脏药物阻滞剂（beta blockers）的靶目标，生物通注)来进行研究。研究人员制备了嵌入β2 肾上腺素受体的nanodiscs，用一种类似药物的小分子化合物来处理这个nanodiscs，由于这种化合物已知可以结合在膜蛋白向膜外的受体结合部位，这种结合会导致β2 肾上腺素受体改变形状并释放原来结合在β2 肾上腺素受体细胞膜内部分的G蛋白。通过同位素标记小分子化合物，研究小组发现在nanodiscs中的膜蛋白进行了同样的步骤。

University of Wisconsin一位化学家表示，这是一个非常cool的技术，应用前景非常广泛，这个技术将有助于解开一大堆未知的膜蛋白的生化行为模式，也将有可能帮助得到膜蛋白的结晶，从而应用X射线晶体衍射学获得在原子水平上的结构图。”

如果真的这样，结构生物学可以得到大发展了。这项技术应用与研究其功能是显而易见的，但不太理解如何用于研究结构，怎样长晶体？希望能尽快找到相关的资料，看看能不能学习一下。

找到出处：

在Science上的原文：

Ring of Hope for Membrane Proteins

SAN FRANCISCO--Among the most important molecules in biology are proteins that wedge themselves into the fatty membranes surrounding cells. As gatekeepers, they detect key compounds outside the cell and determine which should be allowed inside. Studying these receptor molecules in detail is extremely difficult, because they almost always stop working if removed from the cell membrane. But now a team has found a way to put them on display while making it seem as if they've never left home. Almost home. Discs of phospholipid molecules allow researchers to isolate membrane proteins in an environment closely resembling the cell membrane. CREDIT: S. SLIGAR/UIUC At a meeting here last week of the Materials Research Society, Stephen Sligar, a biochemist at the University of Illinois, Urbana-Champaign, reported that he and his colleagues have ensconced individual membrane-bound receptors in lipid-based nanodiscs. Like natural cell membranes, the discs are composed of two back-to-back layers of phospholipid molecules, each sporting water-friendly head groups and long, oily water-repellant tails. To keep the discs flat, the team fashioned a belt of proteins that surround the disc like the seaweed wrapper on a sushi roll. To demonstrate that receptors still function inside the nanodisc, Sligar's graduate student Andrew Leitz turned to a well-known protein: the β2 adrenergic receptor (β2AR), a target of heart drugs called beta blockers. The researchers dosed β2AR-containing discs with a small druglike compound that normally binds to the portion of the receptor facing out of the cell. The binding triggers the β2AR to change shape and release a "G" protein, which in cells binds to the inside end of the receptor. Using radiolabeled compounds, the group found that the receptors in the discs traced the same key steps. "It's a very cool technique," says Robert Hamers, a chemist at the University of Wisconsin, Madison. "I can see all kinds of applications for something like this." Hamers says nanodiscs could shed light on the biochemical behavior of a host of membrane proteins that have escaped detailed understanding. In time the method may also make it possible to crystallize membrane proteins to obtain atomic-level maps of their structure using x-ray crystallography, another long-elusive goal. --ROBERT F. SERVICE Related Sites Sligar Lab home page

关于那次会议的资料：

Nanotechnology for Cracking the Problems of Membrane Proteins: Functional Incorporation of Integral and Embedded Membrane Proteins into Soluble Nanodiscs. Stephen G. Sligar, Dept of Biochemisty, Chemistry and the College of Medicine, University of Illinois-Urbana, Urbana, Illinois.

另外：

Nanotechnology for Cracking the Problems of Membrane Proteins
Stephen G. Sligar
Departments of Biochemistry, Chemistry and the College of Medicine
University of Illinois at Urbana-Champaign and Nanodisc LLC, Urbana, Ilinois 61801

Nanobiotechnology is the marriage of biology with the nanotechnological advances in materials, instrumentation and processing in order to realize a fundamentally new understanding of biological function as well as to visualize and manipulate hierarchical supramolecular assemblies. An important goal is the development and execution of methodologies for the determination of biological structure and function in the 5 nm - 500 nm 'mesoscale' size range, thus providing the important architectural information of specific aggregates of nucleic acids, lipids and proteins which constitute important cellular machinery. We have utilized genetically engineered lipid-protein complexes to understand the reactivity of lipoproteins on surfaces and to stabilize and incorporate single membrane proteins in nanostructured phospholipid bilayers. These resultant supramolecular architectures, termed NanodiscsTM, allow the direct visualization of individual membrane protein structures and measurement of physical properties on single molecules. The ability to directly probe the function of single membrane proteins incorporated into mimics of the natural cellular environment can have an enormous impact on the understanding and control of biological signaling, receptor mediated growth control processes and high throughput screening.