噬菌体434单链阻遏蛋白的高亲和力DNA结合序列的设计和筛选

一． 设计和筛选单链阻遏蛋白的高亲和力DNA结合序列 　　单链阻遏蛋白RRTRES是噬菌体434阻遏蛋白的衍生物，它是噬菌体434阻 遏蛋白的N端DBD(1-69位氨基酸)组成的共价二聚体。这个单链分子有两个DBD，一个是野生型噬菌体434的DBD-R，另一个是突变了的DBD - RTRES，二者用重组接头以头接尾的方式连接起来。在RTRES的α3-螺旋中．1、1、2、5位氨基酸与DNA识别紧密相关，它们分别为T、R、E、S。为了筛选出突变的RTRES的DNA结合位点，设计了核心序列为CATACAAGAAAGNNNNNNTTTATG随机DNA库，通过RRTRES与随机DNA库的体外结合和循环筛选。将筛选到的群体克隆并测序。通过与单链阻遏蛋白RRTRES的亲和力测定，对每一个筛选到的序列进行特性分析。结果表明，当结合位点（上述划线部分）为TTAC或TTCC时为最适操纵区序列。它们与单链阻遏蛋白RRTRES的亲和力很高，Kd值在1-10pM的范围。其中随机部分为TTTACG的操纵区与RRTRES的亲和力最高，Kd值约为lpM;当结合位点为TTAC时，平均Kd值为3pM：当结合位点为 TTCC时，Kd值在5-lOpM之间。天然噬菌体434阻遏蛋白与其操纵区的亲和力的Kd值在nM数量级，与之相比，所筛选操纵区的亲和力明显提高。此外，亲和力大小还受到结合位点两侧的碱基的影响，特别是5'位碱基的影响。 　　表达纯化同源双突变的单链阻遏蛋白RTRESRTRE'根据RTRES的以上识别特一点，设计了一系列新的操纵区序列，它们的共有序列为GTAAGAAARNTTACN，或GGAAGAAARNTTCCN，并测定它们与RTRES RTRE之间的结合特异性。结果表明，它们可被RTRES RTRES特异识别，且亲和力也很高，Kd值在5-40pM之间。其中GTAAGAAAGTTTACG与RTRES RTRES之间结合的Kd值约为5pM。同样，表达了异源双突变的单链阻遏蛋白R*RTRES，然而它与 设计的相关操纵区的亲和力并不很高，Kd值约为lOOpM。利用本工作中的随机筛选和合理设计的原则，得到了新的具有特异性识别和高亲和力的蛋白一DNA相互作用。这个方法可望用于其他DBP的新的结合特异性的筛选。 二． 非同位素的方法筛选单链阻遏蛋白的最佳DNA结合序列初探 　　克隆和表达了带半胱氨酸尾的单链阻遏蛋白，利用已包被了马来酰胺的活性板可以与自由巯基结合的特性，将蛋白固定在活性板表面。体外筛选RTRES RTRES的最佳DNA结合序列，得到了一些与RTRES RTRES结合的序列，但Kd值nM数量级。此方法需进一步优化。

蓝藻应答高 pH 胁迫的亚细胞蛋白质组学研究

蓝藻是迄今地球上发现的最古老、分布最广和最具多样性的光合自养原核生物，其细胞结构简单，具有类似于植物的光合作用，是研究光合作用及其它代谢过程重要的模式生物。由于这类生物起源于远古前寒武纪，但至今依然繁多，在极端寒冷的南北极冰湖和近于沸腾温度的温泉，以及高盐、强碱的极端环境中均有存在,它们在漫长的进化过程中如何应对灾难性环境、针对随时可能遭遇的不同胁迫环境因子形成了怎样的分子适应机制，是近年来倍受关注但仍未诠释的问题之一。由于蓝藻与高等植物叶绿体在进化上密切相关，搞清楚这类生物适应不同胁迫环境因子的分子基础及其作用机制，对从进化的角度理解光合生物与环境相互作用、通过同源性发现作物抗逆育种新靶标，有重要的理论和实践意义。 逆境应答蛋白的表达是细胞对逆境胁迫的主要适应机制之一。在特定的逆境条件下，细胞通常会表达一组蛋白质，用于识别与传递环境胁迫信号、稳定细胞内环境、消除并修复逆境造成的损伤等。因此，逆境应答蛋白的系统鉴定和功能确认，是揭示逆境条件下细胞代谢网络及抗逆性分子机制的关键。单细胞模式蓝藻基因组序列的确定，极大地推动了蓝藻细胞蛋白质组成模式研究，也为系统发掘蓝藻逆境应答蛋白、理解和揭示分子适应机制提供了新的切入点。Synechocystis 6803是第一个完成基因组测序的放氧光合模式生物。由于其具有易培养、可转化、对环境条件变化反应快等优点，以该藻种为材料所展开的逆境应答特别是盐胁迫蛋白质组研究方面已经取得了重要的进展，而对高pH胁迫的蛋白质组研究还鲜有报道。因此，本论文以Synechocystis 6803为材料，从分离纯化的亚细胞组分入手，采用蛋白质组学研究手段，对蓝藻细胞应答高pH胁迫的蛋白质代谢网络进行探讨。利用蔗糖密度离心和水溶性两相分离法相结合的方法，分别获得了对照（pH7.5）和处理（pH11）细胞的质膜、外膜和类囊体膜，并分别构建了包括可溶性蛋白和膜组分的一维和二维蛋白质凝胶电泳图谱。分析结果表明，高pH胁迫下质膜和可溶性蛋白蛋白组分的变化较外膜和类囊体膜蛋白组分更为明显。在考马斯亮兰染色胶上共发现有近110个蛋白点上调或下调表达，其中有82个蛋白点来源于质膜。对质膜蛋白进行的差异荧光标记双向电泳（2-D DIGE）分析结果与考马斯亮兰染色结果基本一致。对质膜上的82个蛋白点进行胶内消化和MALDI-TOF和MALDI-TOF/TOF质谱鉴定，得到了39个不同基因产物，其中25个是上调蛋白，14个是下调蛋白。在这些发生变化的蛋白中，近1/3是ABC型转运蛋白，如3个磷转运蛋白（Sll0679，Sll0683，Sll0684）均在高pH胁迫下明显上调。其它高pH响应蛋白包括参与光合作用（PsaF，Sll0819；CpcA，Sll1578）、呼吸作用（CoxB，Sll0813）以及细胞分裂过程的蛋白（MinD，Sll0289）。还有LexA repressor (Sll1626)和Guanylyl cyclase(Cya2，Sll0646)等起调控作用的蛋白质。此外发现8个高pH胁迫响应蛋白为功能未知的新蛋白。生物信息学预测结果显示，在已鉴定的质膜响应蛋白中有17个蛋白具有信号肽。6个蛋白为具有跨膜域的膜蛋白，其中的3个膜蛋白是首次被证明定位于质膜上，且其表达与高pH胁迫有关。这些研究结果对从分子水平理解蓝藻细胞主动应对高pH胁迫、维护细胞内pH相对稳定机制有重要启示。

Configuration-dependent diffusion can shift the kinetic transition state and barrier height of protein folding

We show that diffusion can play an important role in protein-folding kinetics. We explicitly calculate the diffusion coefficient of protein folding in a lattice model. We found that diffusion typically is configuration- or reaction coordinate-dependent. The diffusion coefficient is found to be decreasing with respect to the progression of folding toward the native state, which is caused by the collapse to a compact state constraining the configurational space for exploration. The configuration- or position-dependent diffusion coefficient has a significant contribution to the kinetics in addition to the thermodynamic free-energy barrier. It effectively changes (increases in this case) the kinetic barrier height as well as the position of the corresponding transition state and therefore modifies the folding kinetic rates as well as the kinetic routes. The resulting folding time, by considering both kinetic diffusion and the thermodynamic folding free-energy profile, thus is slower than the estimation from the thermodynamic free-energy barrier with constant diffusion but is consistent with the results from kinetic simulations. The configuration- or coordinate-dependent diffusion is especially important with respect to fast folding, when there is a small or no free-energy barrier and kinetics is controlled by diffusion.Including the configurational dependence will challenge the transition state theory of protein folding.

Identification and characterization of the AcrR/AcrAB system of a pathogenic Edwardsiella tarda strain

Edwardsielia tarda is one of the leading marine pathogens that can infect a wide range of cultured marine species. In this study, the acrR-acrAB cluster was cloned from TX1, a pathogenic E. tarda strain isolated from diseased fish. AcrR and AcrAB were found to be involved in resistance against acriflavine and methyl viologen, which positively regulate the expression of acrAB. AcrR negatively regulates its own expression and the expression of the acrAB operon, most likely by interacting with a 24-bp operator site that overlaps the putative promoter of acrA (PacrA). The repressive effect of AcrR on PacrA could be relieved by acriflavine, methyl viologen, and ethidium bromide, the presence of each of which enhanced transcription from PacrA. Interruption of the regulated expression of acrR by introducing into TX1 a plasmid that overexpresses acrR affected growth under stress conditions, AI-2 production, and bacterial virulence. In addition, mutational analyses identified a constitutively active AcrR mutant (named N215), which exhibits full repressor activity but is impaired in its ability to interact with the inducer. Overexpression of N215 produced the same kind of but moderately stronger effect on TX1 compared to that produced by overexpression of the wild-type acrR.

Domain analysis of the Edwardsiella tarda ferric uptake regulator

Recent studies have shown that the ferric uptake regulator (Fur) of Edwardsiella tarda (Fur(Et)) shares high sequence identity with the Escherichia coli Fur (Fur(Ec)) at the N-terminal DNA-binding region. In the present study, the functional importance of the C-terminal region of Fur(Et) was investigated. It was found that Fur(Et) bearing deletion of the C-terminal 12 residues still possesses most of the repressor activity, whereas Fur(Et) bearing deletions of the C-terminal 16 and more than 16 residues are severely affected in activity. Domain swapping analyses indicated that the chimeric Fur proteins (Et75Ec73 and Et75Vh74) consisting of the N-terminal 1-75 region of Fur(Et) fused to the C-terminal 76-148 region of Fur(Ec) and the C-terminal 76-149 region of the Vibrio harveyi Fur (Fur(Vh)), respectively, are fully active. C92 of Fur(Ec) and C137 of Fur(Vh), which are functionally essential in Fur(Ec) and Fur(Vh), respectively, are also essential in Et75Ec73 and Et75074, respectively. Further study identified an artificial Fur protein, EtMF54, which is composed of the N-terminal 49 residues of Fur(Et) and five artificial residues. Compared to Fur(Et), EtMF54 possesses partial Fur activity that is iron-dependent. These results (I) indicate that there exist certain functional/structural compatibilities among Fur(Et), Fur(Ec), and Fur(Vh) at the C-terminal region; (ii) provide insights to the potential location of the regulatory ion-binding site of Fur(Et).

Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur(vh)) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and could complement a mutant of Fur(Ec). Like Fur(Ec), Fur(Vh), possesses two cysteine residues at positions 92 and 95, yet unlike Fur(Ec), in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur(Vh) proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur(vh) are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur(Vh) are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur(Vh) is possibly different from that of Fur(Ec); and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur(Vh); and (iii) provided insights into the potential function of the local structure involving C137 and K138.