用嫁接活性位点方法设计新的功能蛋白质

将一个蛋白质的活性位点,嫁接到另一个分子量较小但是稳定的蛋白质（骨架蛋白）上,从而形成一种新的功能蛋白质,这是蛋白质设计中一种很有效的方法,在我们发展的异型自洽系综最优化方法的基础上,结合３Ｄ－模体搜索等工具,实现了这种位点嫁接的设计。并以在卡律蝎毒素分子骨架上嫁接碳酸酐酶Ｂ的Ｚｎ＾２＋结合位点的设计为例进行检验,表明此设计系统是可行的和有效的。     

蛋白质二级结构指定

蛋白质二级结构是指蛋白质骨架结构中有规律重复的构象。由蛋白质原子坐标正确地指定蛋白质二级结构是分析蛋白质结构与功能的基础,二级结构的指定对于蛋白质分类、蛋白质功能模体的发现以及理解蛋白质折叠机制有着重要的作用。并且蛋白质二级结构信息广泛应用到蛋白质分子可视化、蛋白质比对以及蛋白质结构预测中。目前有超过20种蛋白质二级结构指定方法,这些方法大体可以分为两大类:基于氢键和基于几何,不同方法指定结果之间的差异较大。由于尚没有蛋白质二级结构指定方法的综述文献,因此,本文主要介绍和总结已有蛋白质二级结构指定方法。     

源于红树林的新型脂肪酶Lip906生物信息学分析

本研究利用生物信息学在线软件对脂肪酶Lip906的二级结构和模体信息进行预测,同时对其三级结构进行同源建模和模建结果质量评价,预测该蛋白质的活性位点信息,旨在从蛋白质序列特征和分子结构水平理解其在酯类水解过程中的作用。结果表明,模建的Lip906蛋白结构品质较高,具有7段α-螺旋和2组β-折叠结构,是一个典型的α/β类蛋白,表面呈弱负电势分布;Lip906蛋白具有5个不同模体,可能参与不同生化反应或执行不同的功能。这些研究结果对理解Lip906蛋白功能以及配基结合位点定位非常重要,也为脂肪酶Lip906的突变设计提供了理论基础。     

锚蛋白重复序列介导的蛋白质-蛋白质相互作用

锚蛋白重复序列(ANK)是生物体中广泛利用的一种序列模体.ANK模体在ANK结构域中折叠成β₂α₂结构,在空间上则形成L型结构.数目不等的ANK串联起来, 依靠氢键和疏水相互作用,组成紧密、稳定的结构域,并且形成了种类众多但功能各异的ANK蛋白质分子.ANK结构域介导蛋白质与蛋白质的相互作用,它能够和多种配体结合,实现纷繁复杂的生物功能.着重介绍几类结构已知的ANK家族蛋白质分子及复合物的结构特征、生理功能及与疾病的关系.     

线性短模体：介导蛋白质相互作用的新模块

线性短模体是天然无序蛋白实现生物学功能的重要组件.线性短模体具有柔性结构和短小的序列,可以介导瞬时、可逆的蛋白质相互作用,并在发生相互作用时表现出杂泛性.随着实验技术的更新和预测手段的发展,越来越多的线性短模体被发现和重新定义,例如BH3线性短模体.本文重点总结了线性短模体在结构、生物学功能以及进化等方面的特点.对线性短模体功能的研究将为解析细胞信号转导网络、疾病靶标确认、新药发现等领域带来新的思路.     

重组人蛋白质二硫键异构酶活性片段的克隆与表达纯化策略

蛋白质二硫键异构酶(protein disulfide somerase,PDI)在体内或体外均可以非特异地催化其它蛋白质的二硫键的形成、还原和异构化,进而辅助蛋白的折叠和复性。近来发现PDI还具有非ATP依赖的分子伴侣的功能。鉴于PDI具有的重要功能,拟利用PDI来辅助基因工程产品的复性,如包含体蛋白(尤其是含有二硫键的蛋白),这将为基因工程产品的下游纯化复性,开拓新的思路。PDI含有两个硫氧还蛋白(thioredoxin,TRX)同源区a区和a′区,各含有一个CGHC活性位点,与TRX的CGPC位点同源。a区和a′区各具有50％的二硫键异构     

应用突变的断裂蛋白质内含子标记蛋白质N端

蛋白质的特异位点修饰可以帮助了解蛋白质的结构与功能.但是,现有的蛋白质特异位点标记方法种类有限,而且存在局限性,所以有必要开发新的蛋白质特异位点标记方法.以谷胱甘肽-S-转移酶(GST)为研究对象,借助蛋白质反式剪接技术,建立了利用新型断裂蛋白质内含子对蛋白质进行N端标记的新方法.在这个方法中,通过简单的重组表达、标记和纯化得到带有荧光基团的小肽,经过蛋白质反式剪接,荧光基团被标记到蛋白质的N端.初步研究结果显示,标记效率可达到12%.     

真核蛋白质的亚细胞位点预测研究进展

研究真核蛋白质的亚细胞位点是了解真核蛋白质功能,深入研究蛋白质相关信号通路内在机制的基础。同时,可以为了解 疾病发病机制及为新药研发提供帮助。因此,研究真核蛋白质的亚细胞位点意义十分重大。随着基因组测序的完成,真核蛋白质 序列信息增长迅速,为真核蛋白质亚细胞位点的研究提出了更多的挑战。传统的实验法难以满足蛋白质信息量迅速增长的需求。 而采用生物信息学手段处理大规模数据的计算预测方法,可在较短时间内获得大量真核蛋白质亚细胞位点信息,弥补了实验法 的不足。因此,运用计算预测法预测真核蛋白质的亚细胞位点成为生物信息学领域的研究热点之一。本文主要从提取真核蛋白质 的特征信息、计算预测方法及预测效果的评价三个方面,介绍近年来真核蛋白质亚细胞位点预测的研究进展。     

应用N端Cys-free断裂蛋白质内含子标记蛋白质N端

蛋白质的特异位点修饰可以帮助了解蛋白质的结构与功能.但是,现有的蛋白质特异位点标记方法种类有限,而且存在局限性,所以有必要开发新的蛋白质特异位点标记方法．以谷胱甘肽-S-转移酶(GST)为研究对象,借助蛋白质反式剪接技术,建立了利用新型断裂蛋白质内含子对蛋白质进行N 端标记的新方法.在这个方法中,通过简单的重组表达、标记和纯化得到带有荧光基团的小肽,经过蛋白质反式剪接,荧光基团被标记到蛋白质的N 端. 初步研究结果显示,标记效率可达到12 %.     

多肽脯氨酰顺反异构酶与阿尔茨海默病

多肽脯氨酰顺反异构酶(简称Pinl)可专一性地使蛋白质一定区段内磷酸化的丝／苏氨酸模体发生顺反异构,从而改变该蛋白质的构型和功能。阿尔茨海默病中发生特征性改变的蛋白质如微管相关蛋白tau、淀粉样前体蛋白(APP)等都含有丝／苏氨酸模体,它们的活性和功能均受Pinl的变构调节。     

AutoMatch: target-binding protein design and enzyme design by automatic pinpointing potential active sites in available protein scaffolds

Proteins perform their functions mainly via active sites, whereas other parts of the proteins comprise the scaffolds, which support the active sites. One strategy for protein functional design is transplanting active sites, such as catalytic sites for enzyme or binding hot spots for protein-protein interactions, onto a new scaffold. AutoMatch is a new program designed for efficiently elucidating suitable scaffolds and potential sites on the scaffolds. Backrub motions are used to treat backbone flexibility during the design process. A step-by-step checking strategy and cluster-representation examination strategy were developed to solve the large combinatorial problem for the matching of active-site conformations. In addition, a grid-based binding energy scoring method was used to filter the solutions. An enzyme design benchmark and a protein-protein interaction design benchmark were built to test the algorithm. AutoMatch could identify the hot spots in the nonbinding protein and rank them within the top five results for 8 of 10 target-binding protein design cases. In addition, among the 15 enzymes tested, AutoMatch can identify the catalytic active sites in the apoprotein and rank them within the top five results for 13 cases. AutoMatch was also tested for screening scaffold library in designing binding proteins targeting influenza hemagglutinin, HIV gp120, and epidermal growth factor receptor kinase, respectively. AutoMatch, and the two test sets, ActApo and ActFree, are available for noncommercial applications at http://mdl.ipc.pku.edu.cn/cgi-bin/down.cgi.     

Comparison of protein active site structures for functional annotation of proteins and drug design

Rapid and accurate functional assignment of novel proteins is increasing in importance, given the completion of numerous genome sequencing projects and the vastly expanding list of unannotated proteins. Traditionally, global primary-sequence and structure comparisons have been used to determine putative function. These approaches, however, do not emphasize similarities in active site configurations that are fundamental to a protein's activity and highly conserved relative to the global and more variable structural features. The Comparison of Protein Active Site Structures (CPASS) database and software enable the comparison of experimentally identified ligand-binding sites to infer biological function and aid in drug discovery. The CPASS database comprises the ligand-defined active sites identified in the protein data bank, where the CPASS program compares these ligand-defined active sites to determine sequence and structural similarity without maintaining sequence connectivity. CPASS will compare any set of ligand-defined protein active sites, irrespective of the identity of the bound ligand.     

Design principles for chlorophyll-binding sites in helical proteins

The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, and hemes are ubiquitous cofactors of biological catalysis that are involved in a multitude of reactions. One systematic approach for understanding how Nature achieves functional diversity with only this handful of cofactors is by designing de novo simple and robust protein scaffolds with heme and/or (bacterio)chlorophyll [(B)Chls]-binding sites. This strategy is currently mostly implemented for heme-binding proteins. To gain more insight into the factors that determine heme-/(B)Chl-binding selectivity, we explored the geometric parameters of (B)Chl-binding sites in a nonredundant subset of natural (B)Chl protein structures. Comparing our analysis to the study of a nonredundant database of heme-binding helical histidines by Negron et al. (Proteins 2009;74:400-416), we found a preference for the m-rotamer in (B)Chl-binding helical histidines, in contrast to the preferred t-rotamer in heme-binding helical histidines. This may be used for the design of specific heme- or (B)Chl-binding sites in water-soluble helical bundles, because the rotamer type defines the positioning of the bound cofactor with respect to the helix interface and thus the protein-binding site. Consensus sequences for (B)Chl binding were identified by combining a computational and database-derived approach and shown to be significantly different from the consensus sequences recommended by Negron et al. (Proteins 2009;74:400-416) for heme-binding helical proteins. The insights gained in this work on helix- (B)Chls-binding pockets provide useful guidelines for the construction of reasonable (B)Chl-binding protein templates that can be optimized by computational tools.     

Designing and using RNA scaffolds to assemble proteins in vivo

RNA scaffolds are synthetic noncoding RNA molecules with engineered 3D folding harnessed to spatially organize proteins in vivo. Here we provide a protocol to design, express and characterize RNA scaffolds and their cognate proteins within 1 month. The RNA scaffold designs described here are based on either monomeric or multimeric units harboring RNA aptamers as protein docking sites. The scaffolds and proteins are cloned into inducible plasmids and expressed to form functional assemblies. RNA scaffolds find applications in many fields in which in vivo organization of biomolecules is of interest. RNA scaffolds provide extended flexibility compared with DNA or protein scaffolding strategies through programmed modulation of multiple protein stoichiometry and numbers, as well as the proteins' relative distances and spatial orientations. For synthetic biology, RNA scaffolds provide a new platform that can be used to increase yields of sequential metabolic pathways.     

Identification of protein functions from a molecular surface database,eF-site

A bioinformatics method was developed to identify the protein surface around the functional site and to estimate the biochemical function, using a newly constructed molecular surface database named the eF-site (electrostatic surface of Functional site. Molecular surfaces of protein molecules were computed based on the atom coordinates, and the eF-site database was prepared by adding the physical properties on the constructed molecular surfaces. The electrostatic potential on each molecular surface was individually calculated solving the Poisson–Boltzmann equation numerically for the precise continuum model, and the hydrophobicity information of each residue was also included. The eF-site database is accessed by the internet (http://pi.protein.osaka-u.ac.jp/eF-site/). We have prepared four different databases, eF-site/antibody, eF-site/prosite, eF-site/P-site, and eF-site/ActiveSite, corresponding to the antigen binding sites of antibodies with the same orientations, the molecular surfaces for the individual motifs in PROSITE database, the phosphate binding sites, and the active site surfaces for the representatives of the individual protein family, respectively. An algorithm using the clique detection method as an applied graph theory was developed to search of the eF-site database, so as to recognize and discriminate the characteristic molecular surfaces of the proteins. The method identifies the active site having the similar function to those of the known proteins.     

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.     

Protein affinity map of chemical space

Affinity fingerprinting is a quantitative method for mapping chemical space based on binding preferences of compounds for a reference panel of proteins. An effective reference panel of <20 proteins can be empirically selected which shows differential interaction with nearly all compounds. By using this map to iteratively sample the chemical space, identification of active ligands from a library of 30 000 candidate compounds has been accomplished for a wide spectrum of specific protein targets. In each case, <200 compounds were directly assayed against the target. Further, analysis of the fingerprint database suggests a strategy for effective selection of affinity chromatography ligands and scaffolds for combinatorial chemistry. With such a system, the large numbers of potential therapeutic targets emerging from genome research can be categorized according to ligand binding properties, complementing sequence based classification.     

Advancing chemistry and biology through diversity-oriented synthesis of natural product-like libraries

Natural products provide the inspiration for a variety of strategies used in the diversity-oriented synthesis of novel small-molecule libraries. These libraries can be based on core scaffolds from individual natural products, specific substructures found across a class of natural products, or general structural characteristics of natural products. An increasing body of evidence supports the effectiveness of these strategies for identifying new biologically active molecules. Moreover, these efforts have led to significant advances in synthetic organic chemistry. Larger-scale evaluation of these approaches is on the horizon, using screening data that will be made publicly available in the new PubChem database.     

CluSTr: a database of clusters of SWISS-PROT+TrEMBL proteins

The CluSTr (Clusters of SWISS-PROT and TrEMBL proteins) database offers an automatic classification of SWISS-PROT and TrEMBL proteins into groups of related proteins. The clustering is based on analysis of all pairwise comparisons between protein sequences. Analysis has been carried out for different levels of protein similarity, yielding a hierarchical organisation of clusters. The database provides links to InterPro, which integrates information on protein families, domains and functional sites from PROSITE, PRINTS, Pfam and ProDom. Links to the InterPro graphical interface allow users to see at a glance whether proteins from the cluster share particular functional sites. CluSTr also provides cross-references to HSSP and PDB. The database is available for querying and browsing at http://www.ebi.ac.uk/clustr.     

SABER: a computational method for identifying active sites for new reactions

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof‐of‐principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o‐succinyl benzoate synthase (OSBS). Among the highest‐scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L ‐Ala D/L ‐Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified.