SSB蛋白的高效表达及其与ssDNA的相互作用

大肠杆菌单链结合蛋白SSB在DNA复制、重组和修复中起着重要作用。为研究单链结合蛋白SSB的体外生物功能构建了融合蛋白SSB的表达载体并使其高效表达及易于纯化。ssb基因片段是以E.coli K-12基因组为模板经PCR扩增获得,并通过基因的体外拼接成功构建了表达载体pQE30-ssb。重组菌株M15/ pQE30-ssb经过IPTG的诱导表达了蛋白SSB。收集菌体细胞、超声波破碎后离心取上清进行SDS-PAGE分析,结果表明有一与预期分子量（20.6 kD）相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在。利用固定化金属离子（Ni2+）配体亲和层析柱纯化融合蛋白SSB,其纯度达到90％。通过凝胶层析和等离子共振技术对SSB的生物功能进行了系统研究分析。结果表明,SSB蛋白以四聚体形式与单链DNA分子结合,其亲和力常数（KD）为4.79×10-7 M。     

腾冲嗜热菌单链 DNA 结合蛋白 SSB2 和 SSB3 新的单链 DNA 结合特性

摘要：【目的】揭示腾冲嗜热菌中两个单链DNA结合蛋白SSB2和SSB3的全新的底物结合功能及其不同的体内表达模式。【方法】利用腾冲嗜热菌复制起始位点附近的长度较短的单链DNA为底物,采用非变性聚丙烯酰胺凝胶电泳及western blot方法,研究SSB2和SSB3体外单链DNA结合特征和体内表达模式。【结果】SSB2 与35nt的复制起始区单链DNA（ssDNA）结合, 形成单个SSB2-DNA复合物;当与59 nt ssDNA结合时,可以随着蛋白浓度的递增形成一个或两个SSB2-DNA复合物;而与70n     

天花粉蛋白与膜脂相互作用的研究

天花粉蛋白（ＴＣＳ）在≥２．２２μｍｏｌ／Ｌ时能引起大豆磷脂脂质体内含物释放,Ｃａ＾２＋对这种释放有一定的促进作用,低ＰＨ值也能促进ＴＣＳ与脂质体的作用;将ＴＣＳ与细细胞一起保温;当ＴＣＳ终浓度达１４．７μｍｏｌ／Ｌ时能损伤红细胞膜,产生溶血作用;ＴＣＳ还能作用于红细胞血影膜,改变其脂双层的不对称性。     

细胞膜蛋白与细胞骨架蛋白相互作用研究进展

细胞膜蛋白与胞浆骨架蛋白的相互作用对于维持细胞正常形态,细胞粘附与信号传导有重要作用,含有4.1/JEF结构域的蛋白4.1超家族与含有PDZ结构域的MAGUK蛋白家族能结合多种膜蛋白胞内区与胞浆蛋白,在膜蛋白与胞浆蛋白之间建立联系,对于细胞、细胞-细胞间连接的正常结构与功能的维持有着重要作用。     

微管相关蛋白tau与朊蛋白的相互作用

微管相关蛋白tau参与了许多神经退行性疾病的发生, 其中包括一些人类可传播性海绵状脑病. 为了探讨tau与朊蛋白(PrP)之间可能存在的关系, 首先通过GST pull-down和免疫共沉淀等技术发现重组tau蛋白可通过微管结合区与来源于正常叙利亚仓鼠脑组织中的正常细胞膜朊蛋白(PrP^C)和羊瘙痒因子263K感染仓鼠脑组织的异常朊蛋白(PrPSc)相结合. 利用免疫共沉淀实验发现在正常和羊瘙痒因子感染的仓鼠脑组织中存在tau蛋白与PrPC和PrPSc的相互作用, 并且利用激光共聚焦方法检测到PrP和tau蛋白在CHO细胞内具有共定位的关系. 为了确定PrP与tau蛋白相互作用的部位, 构建了不同区域的PrP片段, 从而证明PrP与tau蛋白相互作用的区域位于PrP的N端序列(23~91 aa). PrP与tau蛋白分子间相互作用的直接实验证据提示tau蛋白可能参与PrP的正常生理功能以及朊病毒病的病理过程.     

细胞因子与膜受体蛋白相互作用的研究方法

细胞因子与膜受体的相互作用是生命活动中的重要环节。癌症、自身免疫疾病、代谢紊乱等疾病的发生都和细胞因子失调有着密切的关系,研究细胞因子和受体的相互作用成为治疗上述疾病的基石。本文综述了现今常用的研究方法,包括从最经典的同位素标记直接检测受体与配体的结合,到生化通路下游信号检测,再到生物物理高通量检测方法。     

LIM蛋白KyoT2与人类紧密连接蛋白2的相互作用

KyoT是一种LIM结构域蛋白,是以RBP－J为诱饵蛋白通过酵母双杂交系统得到的新分子,KyoT2可以抑制RBP－J介导的转录,以KyoT2为诱饵蛋白,通过酵母双杂交筛选得到了人类紧密连接蛋白2（ZO－2）,的一种新的剪接体ZO－2－i3,序列分析表明,新的剪接体有19个外显子,与已发表序列比较,见ZO－2－i3分子的激酶后区发生了改变,为排除酵母双杂交实验的假阳性,实验首先在酵母中验证KyoT2与ZO－2-i3的体外直接相互作用并得到阳性结果,进而在大肠杆菌中原核表达纯化带有His标签的KyoT2蛋白,使用抗His标签的抗体,通过GST pull-down assay验证KyoT2与ZO－2－i3的体外直接相互作用,也获得阳性结果,并通过酵母实验初步确定了其作用位点,即KyoT2通过LIM2结构域与ZO－2-i3相互作用,本实验验证了KyoT2与Z－2－i3的相互作用,并初步确定其相互作用位点,对探讨KyoT的功能具有重要意义。     

DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.     

MBP-MalFGK2相互作用动力学:ABC转运蛋白的模型研究

本文主要描述了麦芽糖结合蛋白(MBP)和属于ATP结合盒式蛋白(ABC)家族的麦芽糖转运蛋白复合物MalFGK2的相互作用。通过基因、结构和生化分析可知,MBP和MalFGK2以不同构象进行相互作用。在这个转运系统中,MBP与麦芽糖结合,并与MalFGK2发生相互作用,从而将麦芽糖从胞外转运至胞内,但由于MBP和MalFGK2都有多种构象,所以它们的相互作用很复杂。相互作用机理模型最重要的特点是结合配体的MBP,通过稳定MalFGK2的高能量构象来启动依赖ATP的麦芽糖转运过程。麦芽糖转运蛋白机理模型表明,ABC型转运系统利用外周结合蛋白,其转运过程基本上是不可逆的。     

蛋白-蛋白相互作用——复合体与共享组成物

蛋白相互作用网络代表了一种理解细胞过程的观点.最近的实验利用高产率质谱分析得到了生理相关多蛋白复合体的多套大量数据,提出了基于一个基本的包括两个部分的图表模式(这种模式优于现在的网络作用模式)的数据系统统一陈述.该陈述允许对在超过一个复合体之间的蛋白质联系进行重要性的评估,以及对在网络作用被视为构成共享组成物的蛋白复合体时发生的高水平组织进行定位.这个陈述同样允许应用复杂的MinMaxCut图表簇算法来决定网络作用中相关蛋白单元.从统计学上看,从在蛋白-蛋白反应和复合体-复合体反应里簇的解释中使用GO(Gene Ontology)中的词组这一点说明了这个方法对构成假设未定性蛋白复合体的组成物或者蛋白复合体之间的未定性关系有用处.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

Crystal Structure of an Unusual Single-Stranded DNA-Binding Protein Encoded by Staphylococcal Cassette Chromosome Elements

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Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a processive 5′-3′ single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg^2 + or Mn^2 + and inhibited by Cd^2 +, suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays. Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interaction between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.     

A Single Residue Determines the Cooperative Binding Property of a Primosomal DNA Replication Protein,PriB, to Single-Stranded DNA

PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)₂₅, and (StPriB)₂₅ are proposed.     

Identification and Characterization of the Single-Stranded DNA-Binding Protein of Bacteriophage P1

The genome of bacteriophage P1 harbors a gene coding for a 162-amino-acid protein which shows 66% amino acid sequence identity to the Escherichia coli single-stranded DNA-binding protein (SSB). The expression of the P1 gene is tightly regulated by P1 immunity proteins. It is completely repressed during lysogenic growth and only weakly expressed during lytic growth, as assayed by an ssb-P1/lacZ fusion construct. When cloned on an intermediate-copy-number plasmid, the P1 gene is able to suppress the temperature-sensitive defect of an E. coli ssb mutant, indicating that the two proteins are functionally interchangeable. Many bacteriophages and conjugative plasmids do not rely on the SSB protein provided by their host organism but code for their own SSB proteins. However, the close relationship between SSB-P1 and the SSB protein of the P1 host, E. coli, raises questions about the functional significance of the phage protein.     

Mutagenesis of the Agrobacterium VirE2 Single-Stranded DNA-Binding Protein Identifies Regions Required for Self-Association and Interaction with VirE1 and a Permissive Site for Hybrid Protein Construction

The VirE2 single-stranded DNA-binding protein (SSB) of Agrobacterium tumefaciens is required for delivery of T-DNA to the nuclei of susceptible plant cells. By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1. VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers. By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1. These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1. The capacity of VirE2 mutants to interact with full-length VirE2 in the yeast Saccharomyces cerevisiae correlated with the abundance of the mutant proteins in A. tumefaciens, suggesting that VirE2 is stabilized by homomultimerization in the bacterium. We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2. A PvirB::virE2 construct yielded functional VirE2 protein as defined by complementation of a virE2 null mutation. By contrast, PvirE or Plac promoter constructs yielded functional VirE2 only if virE1 was coexpressed with virE2. Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function. However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in A. tumefaciens, and (iv) restore wild-type virulence to a virE2 null mutant. We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest for delivery across kingdom boundaries.     

Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities

Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.     

Diffusion of Human Replication Protein A along Single-Stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D₁ ~ 5000 nt² s^− 1 at 37 °C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.