Predicting protein interaction interfaces from protein sequences: Case studies of subtilisin and phycocyanin

Identification of protein interaction interfaces is very important for understanding the molecular mechanisms underlying biological phenomena. Here, we present a novel method for predicting protein interaction interfaces from sequences by using PAM matrix (PIFPAM). Sequence alignments for interacting proteins were constructed and parsed into segments using sliding windows. By calculating distance matrix for each segment, the correlation coefficients between segments were estimated. The interaction interfaces were predicted by extracting highly correlated segment pairs from the correlation map. The predictions achieved an accuracy 0.41-0.71 for eight intraprotein interaction examples, and 0.07-0.60 for four interprotein interaction examples. Compared with three previously published methods, PIFPAM predicted more contacting site pairs for 11 out of the 12 example proteins, and predicted at least 34% more contacting site pairs for eight proteins of them. The factors affecting the predictions were also analyzed. Since PIFPAM uses only the alignments of the two interacting proteins as input, it is especially useful when no three-dimensional protein structure data are available.

Drug-protein interaction studied by technique of microdialysis combined with high performance liquid chromatography

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines the method of microdialysis combined with high-performance liquid chromatography (HPLC) that has been developed;by ours to study such interactions, in which the microdialysis was applied to sample the free drug in the mixed solution of drug with protein, and HPLC to quantify the concentration of free drug in the microdialysate. This technique has successfully been used for determining various types of binding interactions between the low affinity drugs, high affinity drugs and enantiomers to HSA. For the case of competitive binding of two drugs to a protein in solution, a displacement equation has been derived and examined with four nonsteroidal anti-inflammatory drugs and HSA as model drugs and protein, respectively. Microdialysis with HPLC was adopted to determine simultaneously the free solute and displacing agent in drug-protein solutions. The method is able to locate the binding site and determine affinity constants even up to 10(7) L/mol accurately.

Micro-systems for Optical Protein-Chip

Protein-Chip as micro-assays for the determination of protein interaction, the analysis, the identification and the purification of proteins has large potential applications. The Optical Protein-Chip is able to detect the multi-interaction of proteins and multi-bio-activities of molecules directly and simultaneously with no labeling. The chip is a small matrix on solid substrate containing multi-micro-area prepared by microfabrication with photolithography or soft lithography for surface patterning, and processed with surface modification which includes the physical, chemical, and bio-chemical modifications, etc. The ligand immobilization, such as protein immobilization, especially the oriented immobilization with low steric hindrance and high bio-specific binding activity between ligand and receptor is used to form a sensing surface. Each area of the pattern is corresponding to only one bioactivity. The interval between the areas is non-bioactive and optically extinctive. The affinity between proteins is used to realize non-labeling microassays for the determination of protein identification and protein interaction. The sampling of the chip is non-disturbing, performed with imaging ellipsometry and image processing on a database of proteins.

白皮松花粉管发育过程中RNA、蛋白质和多糖类物质的合成及其动态变化

花粉管是有花植物受精过程中雄性生殖单位的载体，它同根毛、真菌菌丝一样，具有典型的极性顶端生长模式。裸子植物花粉与被子植物相比，具有萌发时间较长，生长缓慢等特点。但是目前人们对于裸子植物花粉萌发和花粉管生长的机理还不清楚。本文以裸子植物白皮松（Pinus bungeana）的花粉为材料，采用细胞学和生理生化方法，包括应用普通光学显微镜、荧光显微镜、激光扫描共聚焦显微镜、显微红外光谱（FTIR）和透射电镜（TEM）等技术，对其花粉萌发和花粉管生长过程进行了较为系统的研究，旨在进一步揭示裸子植物花粉管发育的调控机制。 本论文首先研究了外源Ca2+ 和3种调钙药物（A23187、EGTA、TMB8）对白皮松花粉萌发和花粉管生长的影响。结果表明，在离体培养条件下，高浓度的Ca2+（1%）能完全抑制白皮松花粉的萌发，低浓度的Ca2+ 则影响不大，而花粉萌发和花粉管生长的最适Ca2+ 浓度为0.01%。用Ca2+ 载体A23187、Ca2+ 螯合剂EGTA和钙通道阻滞剂TMB8分别处理花粉后，花粉萌发和花粉管生长均受到抑制。另外，用 Ca2+ 荧光探针 Fluo-3AM标记，对Ca2+ 的分布变化进行了观察，发现在花粉萌发的初期，Ca2+ 向萌发孔聚集。在正常生长的花粉管中Ca2+ 呈梯度分布，顶端荧光最强。与对照相比，A23187处理后花粉粒中荧光增强，而EGTA和TMB8处理的花粉粒中荧光强度均减弱。并且这3种调钙药物还破坏了花粉管顶端的Ca2+ 浓度梯度，最终导致花粉管的生长受阻。 花粉萌发和花粉管的生长依赖于RNA和蛋白质的不断合成。在放线菌素D的存在下，花粉萌发基本不受影响，但花粉管的生长速度下降，花粉管中RNA含量也减少。而经过放线菌酮处理后，花粉萌发和花粉管生长均受到抑制，花粉管中蛋白质含量降低，同时花粉管顶端显著膨大。通过SDS-PAGE的结果表明，花粉粒萌发前后蛋白质图谱有明显差异。FTIR光谱分析表明，两种抑制剂处理均导致花粉管壁的化学组成发生了变化，例如蛋白质和饱和酯含量减少，而羧酸的含量增加。此外，由放线菌酮和放线菌素D处理后，花粉管的超微结构也发生了明显变化，其中特别是花粉管顶端的分泌系统遭到严重破坏。 纤维素的正常合成对于白皮松花粉管的生长是必需的。在正常培养基中添加纤维素生物合成抑制剂2，6-二氯苯腈（DCB）后，花粉萌发几乎不受影响，但是花粉管的形态发生异常，生长速率降低。DCB处理还导致花粉管壁中纤维素含量下降，而胼胝质在花粉管顶端积累。用识别酯化果胶的JIM7和识别酸性果胶的JIM5对离体培养的白皮松花粉管进行标记后，发现果胶成分呈异常分布图式。FTIR光谱分析结果表明花粉管细胞壁中蛋白质、羧酸以及饱和酯含量增加。同时，在电镜下观察发现，花粉管细胞壁顶端呈现不均匀加厚，其中主要的细胞器，如高尔基体和线粒体等膜结构均遭到破坏。 上述结果说明，白皮松成熟花粉粒中已含有花粉萌发和花粉管早期生长所必需的Ca2+ 和RNA，但是在花粉管的后续伸长过程中仍需要外源Ca2+ 的参与以及新RNA、蛋白质的不断合成。与被子植物不同，裸子植物花粉萌发的启动也需要新蛋白的合成。尽管在花粉管中纤维素的含量很低，但是对于细胞壁的构建、花粉管的正常形态的维持起着关键作用。

DnaJ-like protein gene sll1384 is involved in phototaxis in Synechocystis sp PCC 6803

In Synechocystis sp. PCC 6803, gene sll1384 encodes a protein with a DnaJ domain at its N-terminal portion and a TPR domain at the C-terminal portion. An sll1384 mutant shows no difference from the wild type in adaptation to different temperatures, but almost completely loses its capability of phototactic movement. After complementation with sll1384, the mutant regains the phototaxis. As shown with electron microscopy, on the cell surface, mutant cells have pili that appear to be the same as that of the wild type. Also, the transformation efficiency remains unchanged in the mutant. It is postulated that Sll1384 regulates phototaxis of Synechocystis through protein-protein interaction. It is the first DnaJ-like protein gene identified in a cyanobacterium for a role in phototaxis.

Recent advances in the study of biomolecular interactions by capillary electrophoresis

Capillary electrophoresis (CE) has been abundantly used in the study of molecular interactions owing to such advantages as short analysis time, low sample size requirement, high separation efficiency, and flexible applications. The focus of this paper is to 2 review recent studies and advances (mainly from 1998 to now) in biomolecular interactions using CE. Five CE modes: zone migration CE, affinity CE, frontal analysis (FA), Hummel-Dreyer (HD) and vacancy peak (VP) are cited and compared. Quantitative aspects of the thermodynamics and kinetics of biomolecular interaction are reviewed. Several biomolecular binding systems, including protein-protein (polypeptide), protein-DNA (RNA), protein(polypeptide)-carbohydrate, protein-small molecule, DNA-small molecule, small molecule-small molecule, have been well characterized by CE. CE is shown to be a powerful tool for the determination of the binding parameters of various bioaffinity interactions.

Study of competitive binding of enantiomers to protein by affinity capillary electrochromatography

Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule. (C) 2002 Elsevier Science B.V. All rights reserved.

Uncovering the rules for protein-protein interactions from yeast genomic data

Identifying protein-protein interactions is crucial for understanding cellular functions. Genomic data provides opportunities and challenges in identifying these interactions. We uncover the rules for predicting protein-protein interactions using a frequent pattern tree (FPT) approach modified to generate a minimum set of rules (mFPT), with rule attributes constructed from the interaction features of the yeast genomic data. The mFPT prediction accuracy is benchmarked against other commonly used methods such as Bayesian networks and logistic regressions under various statistical measures. Our study indicates that mFPT outranks other methods in predicting the protein-protein interactions for the database used. We predict a new protein-protein interaction complex whose biological function is related to premRNA splicing and new protein-protein interactions within existing complexes based on the rules generated.

Electrostatic assembly of protein lysozyme on DNA visualized by atomic force microscopy

In the present work, atomic force microscopy (AFM) has been used to study the assembly of protein lysozyme on DNA molecule. Based on the electrostatic interaction, the positively charged lysozyme can easily bind onto the negatively charged DNA molecule surface. The protein molecules appear as globular objects on the DNA scaffold, which are distinguishable in the AFM images. At the same time, lysozyme molecules can be assembled onto DNA as dense or sporadic pattern by varying the protein concentration. This work may provide fundamental aspects for building protein nanostructures and studying of DNA-protein interaction.

Refining near-native protein-protein docking decoys by local resampling and energy minimization

How to refine a near-native structure to make it closer to its native conformation is an unsolved problem in protein-structure and protein-protein complex-structure prediction. In this article, we first test several scoring functions for selecting locally resampled near-native protein-protein docking conformations and then propose a computationally efficient protocol for structure refinement via local resampling and energy minimization. The proposed method employs a statistical energy function based on a Distance-scaled Ideal-gas REference state (DFIRE) as an initial filter and an empirical energy function EMPIRE (EMpirical Protein-InteRaction Energy) for optimization and re-ranking. Significant improvement of final top-1 ranked structures over initial near-native structures is observed in the ZDOCK 2.3 decoy set for Benchmark 1.0 (74% whose global rmsd reduced by 0.5 angstrom or more and only 7% increased by 0.5 angstrom or more). Less significant improvement is observed for Benchmark 2.0 (38% versus 33%). Possible reasons are discussed.

Biosensor with Total Internal reflection Imaging Ellipsometry

In order to monitor multiple protein reaction processes simultaneously, a biosensor based on imaging ellipsometry operated in the total internal reflection mode is proposed. It could be realised as an automatic analysis for protein interaction processes with real-time label-free method. Its principle and methodology as well as a demonstration for its applications are presented.

Development Of Biosensor Based On Imaging Ellipsometry

The concept of biosensor based on imaging ellipsometry was proposed ten years ago. Its principle and the methodology as well as some solutions to problems which have to be faced during the development are mentioned. Its properties of phase sensitive, high throughput and fast sampling, as well as label-free, sensitivity better than 1 ng/ml for Immunoglobulin G, and real-time analysis for protein interaction process, etc. provide a potential for applications in biomedicine field. The recent biosensing development with total internal reflection imaging ellipsometry is presented also. [GRAPHICS] An example of 48 protein arrays in matrix. (C) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Development of Biosensor with Imaging Ellipsometry

The concept of biosensor with imaging ellipsometry was proposed about ten years ago. It has become an automatic analysis technique for protein detection with merits of label-free, multi-protein analysis, and real-time analysis for protein interaction process, etc. Its principle, andrelated technique units, such as micro-array, micro-fluidic and bio-molecule interaction cell, sampling unit and calibration for quantitative detection as well as its applications in biomedicine field are presented here.

三叶木通花发育相关基因的结构、功能和进化研究

花是被子植物最关键的创新（innovation）性状。在被子植物的不同类群中，其形态多种多样，尤其以基部真双子叶植物的花形态最为丰富。大量的系统发育分析表明，在核心真双子叶植物起源之前，几个与花发育相关的MADS-box基因亚家族均发生了大尺度的基因重复事件。因此，在被子植物的不同物种中，花发育相关基因的组成并不相同，并且它们经历了不同的进化历史，这意味着这些基因可能以不同的方式调控花的发育。基部真双子叶植物，作为基部被子植物和核心真双子叶植物之间的过渡类群，对于我们理解被子植物花的进化，揭示核心真双子叶植物花的起源以及基部真双子叶植物花多样性分化的分子机制非常重要。本文以基部真双子叶植物三叶木通为研究材料，着重进行了以下研究工作： 1. 花器官发生过程的观察。三叶木通的花为雌雄同序的单性花。而且，根据成熟花的形态，三叶木通的雌花和雄花都只有一轮花被器官，即三个花瓣状的萼片。扫描电镜的观察结果表明：1）在花器官的发生和发育过程中，在萼片和雄蕊原基之间，确实没有花瓣原基或另一轮萼片原基发生。2）雌花和雄花都是以两性花的方式发生发育的。3）单性花是由于在花发育的最后阶段，雌花中雄蕊或者雄花中心皮的退化而产生的。 2. 花发育相关基因的克隆。应用5’/3’ RACE的方法，我们从三叶木通不同发育阶段的混合花芽中共分离到九个与花发育相关的MADS-box基因： AktFL1、AktFL2、AktAP3_1、AktAP3_2、AktAP3_3、AktPI、AktAG1、AktAG2和AktSEP3。 3. A类MADS-box基因的进化。由于A类基因在进化过程中序列结构的改变，再加上取样的限制，使得A类基因间的进化历史一直不能被很好的理解。因此，本文对A类基因的研究从构建该基因亚家族的系统发育树开始。主要结果如下：1）通过扩大在基部真双子叶植物和被子植物其它重要类群的取样，我们的系统发育树基本上反映了现存被子植物的系统发育关系。2）核心真双子叶植物的A类基因由三个分支组成：euFUL、euAP1和AGL79，它们是通过发生在核心真双子叶植物起源之前的两次几乎同时的基因重复事件产生的。3）在基部真双子叶植物中，山龙眼目、毛茛目和黄杨科的A类基因各形成一支。而且，在这些类群内，发生了多次小尺度的独立的基因重复事件。4）来自单子叶植物的FUL-like基因明显地构成一个单系，并且包括三个分支：OsAMDS14、OsMADS15和OsMADS18。它们是由于两次不连续的基因重复事件产生的。5）不同类型的A类基因产物在C末端拥有不同的保守基元。6）从基因组结构上看，所有的A类基因都拥有八个外显子和七个内含子。7）通过对三叶木通中两个FUL-like型基因（AktFL1和AktFL2）表达式样的观察，我们发现它们在叶原基和发育早期的花原基以及发育着的花器官中都有表达。此外，A类基因表达式样的进化分析结果表明被子植物中该类基因的祖先可能具有广泛的功能，既在营养器官中表达又在生殖器官中表达 。 4. B类基因表达式样的保守性和多样性。通过对B类基因的系统发育和表达式样分析，得到以下结果：1）三叶木通中的三个paleoAP3基因是通过两次基因重复事件产生的。2）在木通科或木通属内，PI型基因并没有发生基因重复事件。3）RT-PCR结果表明，AktAP3_1在雌花中的表达量比雄花中高，而AktAP3_2则在雄花中的表达量比雌花中高。AktAP3_3和AktPI在雌花和雄花中的表达水平相似。4）原位杂交分析显示这些基因在发育着的雄蕊和心皮中表达。此外，AktAP3_3和AktPI还在萼片中表达，可能参与花瓣状萼片的发育。 5. 三叶木通C/D和E类基因的序列结构和表达分析。通过序列结构分析，我们发现，与其它被子植物AG同源基因编码的MADS-domain蛋白一样，AktAG1和AktAG2在MADS结构域的N末端都拥有一段氨基酸序列的延伸，AktAG1为20个氨基酸;AktAG2为7个氨基酸。原位杂交分析表明AktAG1和AktAG2主要在发育着的雄蕊和心皮中表达，说明它们具有决定生殖器官发育这一保守的功能。 AktSEP3属于AGL9型的E类基因。该基因在所有花器官中都有表达，说明和其它被子植物的E类基因一样，AktSEP3在三叶木通中对于所有花器官的发育都是必需的。 6. 各类MADS-domain蛋白间的相互作用。在前面工作的基础上，我们首次对三叶木通中上述MADS-domain蛋白间的作用方式进行了研究。酵母双杂交结果表明：1）AktSEP3的C末端具有转录激活功能。2）三个AktAP3蛋白与AktPI蛋白都能够形成异源二聚体，但是它们之间的作用能力并不相同。3）AktSEP3蛋白可以与AktFL1、AktPI、AktAG1和AktAG2形成异源二聚体，充分体现了E类基因产物作用式样的保守性。4）AktFL1与AktPI、AktSEP3和AktAG2也能形成异源二聚体，这与核心真双子叶植物的euFUL型蛋白在作用式样上是非常相似的。 综合以上结果，我们探讨了三叶木通花发育的分子机制。在三叶木通的三轮花器官中，与拟南芥等模式植物相似的是：E类（AktSEP3）基因在每一轮花器官中都起作用;此外，A类（AktFL1）和B类（AktAP3_3和AktPI）基因在花瓣状的萼片中有不同程度的表达，类似于拟南芥的第二轮;B类（AktAP3_1、AktAP3_2、AktAP3_3和AktPI）和C/D类（AktAG1和AktAG2）基因在雄蕊的发育过程中起作用;C/D类（AktAG1和AktAG2）基因对心皮的发育起作用。与拟 南芥等模式植物不同的是：1）虽然原位杂交分析表明，AktFL1、AktAP3_3、AktPI和AktSEP3都在花瓣状的萼片中有 不同程度的表达，但是它们的蛋白质产物AktFL1与AktSEP3和AktAP3_3与AktPI都只能形成较弱的异源二聚体。而 且，根据我们的研究结果，在三叶木通中没有找到euAP1型的A类基因，只有两个FUL-like型的A类基因。它们的功能 与核心真双子叶植物中的euFUL型基因相似。因此，AktFL1很可能与其它调控因子共同作用负责花分生组织的形成;AktFL1/AktAG2则可能在花发育的后期起作用。那么，三叶木通花瓣状萼片的发育是否需要AktFL1/AktSEP3和 AktAP3_3/AktPI的参与，还是另有其它转录因子的参与，仍然需要更深入的研究。2）虽然在三叶木通中，雄蕊的发 育同样需要B、C/D和E类基因的参与，但是由于小尺度的基因重复事件，在该物种中只拥有三个paleoAP3型基因，而没有euAP3型基因。而且，由于复制拷贝间的亚功能化，AktAP3_1/AktPI主要参与雌花的发育过程;而AktAP3_2/AktPI主要参与雄花的发育过程。

金粟兰花发育相关基因的结构、功能和进化研究

花是被子植物区别于其它植物大类群的最重要的特征，其形态多种多样。花的发育取决于一个复杂的涉及到多个基因和过程的调控体系，因此花的起源和多样化过程实际上可以理解为这个调控体系的进化过程。在被子植物的不同物种中，花发育相关基因的组成并不相同，且经历了不同的进化历史，这意味着这些基因可能以不同的方式调控花的发育。相对于核心真双子叶植物相对稳定的花形态结构而言，基部被子植物的花具有丰富的多样性。因此，对基部被子植物花发育相关基因的研究对于我们理解被子植物花的进化非常重要。 金粟兰科（Chloranthaceae）是基部被子植物的代表类群之一。与研究得比较深入的模式植物相比，花被、雄蕊或雌蕊的缺失，使得该科植物的花比较简单。因此对该科植物中花发育基因的研究不仅有助于揭示花的起源以及花多样性分化的分子机制，还将为认识花部构造简单化的机制提供资料。本文以金粟兰（Chloranthus spicatus）为实验材料，取得了以下研究结果： 1.花发育相关基因的克隆 应用5’/3’RACE的方法，我们从金粟兰不同发育阶段的混合花芽中克隆到了与花发育相关的MADS-box基因：CsPI、CsAG1和CsAG2。 2. 两个A类MADS-box基因表达式样的对比分析 在营养分生组织向生殖分生组织的转变中，花原基的形成，以及随后雄蕊、心皮、花粉、胚珠和胚囊的发育中，CsAP1-1和 CsAP1-2基因均表达。唯一不同之处在于，在花发育成熟期，CsAP1-1在外珠被也有表达，而CsAP1-2在外珠被处没有表达，而只在内珠被处表达。这一结果反映了基因重复事件发生后，两个基因在功能上也有了一些分化。 3. B类基因功能的保守性和多样性 通过转基因实验和蛋白质相互作用研究对B类基因的功能和作用方式进行了研究，得到以下结果：1）金粟兰CsAP3基因的C末端的点突变所造成的paleoAP3基元的部分缺失对该基因的功能没有决定性的影响;2）金粟兰paleoAP3型基因CsAP3所编码蛋白的C末端以及paleoAP3基元，与拟南芥euAP3型基因AtAP3所编码蛋白的C末端以及euAP3基元没有功能上的不同;3）金粟兰paleoAP3型基因CsAP3与拟南芥euAP3型基因AtAP3 的主要功能存在一定差异，前者主要参与雄蕊形成，而后者既参与雄蕊的形成也参与花瓣的形成; 4）CsPI基因所编码的蛋白可以与AP3类蛋白相互作用进而影响花瓣的形成，因此该基因在功能上是保守的。 4. 金粟兰CsAG1基因的序列结构和功能分析 通过序列结构分析发现，CsAG1属于C类基因，具有保守的AG I基元和AG II基元。过量表达实验分析表明CsAG1的功能与A类基因的功能是相拮抗的。 5. 各类MADS-domain蛋白间的相互作用 在前面工作的基础上，我们首次对金粟兰中各类MADS-domain蛋白间的作用方式进行了研究。酵母双杂交结果表明：1）C末端的完整性对于MADS-domain蛋白二聚体的形成没有影响; 2）去掉M区的CsAP3蛋白与CsPI蛋白都能够形成异源二聚体，同时它们又可以各自形成同源二聚体; 3）E类蛋白既可以和A类或C类基因产物相互作用，也可以同AP3和PI型蛋白相互作用，充分体现了E类基因产物作用式样的保守性; 4）金粟兰中，FUL-like型基因所编码的蛋白CsAP1-1与CsSEP3和CsAG1也能形成异源二聚体，这与核心真双子叶植物的euFUL型蛋白在作用式样上是非常相似的。然而，金粟兰CsAP1-1蛋白不能形成同源二聚体。 综合以上结果发现，在无花被的金粟兰中，仍然存在着A、B、C/D、E类花发育相关的基因。这些基因的功能与核心真双子叶植物中同类基因的相比，有些是保守的，比如CsPI基因可以参与花被的形成;但也有一些是不同的，比如CsAP3基因主要参与雄蕊形成而非花被形成过程。由此可以看出被子植物花器官的发育是一个非常复杂的调控过程，不同植物中的调控机理及进化历程可能是不同的。