Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites

The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry. The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 degrees C it is 1.45microM and increases with temperature. To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP. The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments. Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 degrees C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5nM and 6.4nM, corresponding to Gibbs energies of -49kJmol(-1) and -46kJmol(-1), respectively. Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites. In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AP-1 and ATF/CREB sites, and correspondingly the entropies of association, are very different. bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AP-1 complex incorporates significantly more water molecules than the ATF/CREB complex.     

鉴定免疫细胞中DNA疫苗结合蛋白

为研究DNA疫苗从细胞质到细胞核的过程,对免疫细胞中DNA疫苗的结合蛋白进行初步鉴定。 提取小鼠脾脏免疫细胞的细胞质蛋白,将细胞质蛋白分别与 DNA 疫苗 pVAX-OVA 和空载体 pVAX 共孵育,孵育后首先由琼脂糖凝胶电泳分离与DNA结合的蛋白,然后通过SDS-PAGE进行分离和纯化,最后应用质谱技术分析其蛋白组分。质谱结果初步鉴定了免疫细胞中pVAX-OVA 结合的蛋白有IQ motif containing F4 等。免疫细胞中与 pVAX 结合的蛋白有Foxl2,SUV420H2 和 gamma actin 等。Foxl2具有核定位序列,DNA结合区域和与核转运蛋白相互作用的特点,可能对DNA疫苗的进入具有促进作用。这些蛋白质是否可以影响DNA疫苗有效进入细胞核进行基因表达需要进一步研究。     

Time resolved fluorescence of bacteriophage Pfl DNA binding protein and its complex with DNA

The DNA binding protein of the filamentous bacteriophage Pfl exhibits fluorescence from a single tryptophan residue. The location of the emission maximum at 340 nm ist quite common for proteins, but the single lifetime of 7.8 ns is one of the longest yet reported. Protein fluorescence is quenched more efficiently by Cs⁺ than by I^-; the Trp is located in a partially exposed pocket, in the vicinity of a negative charge.In the native complex of the binding protein with Pfl DNA the fluorescence emission maximum is at 330 nm, indicating a more apolar environment for Trp 14. The native nucleoprotein complex exhibits a similar fluorescence lifetime (6.5 ns) and an approximately equal fluorescence yield, indicating the absence of Trp-DNA stacking. The tryptophan in the complex is virtually inaccessible to ionic quenchers, and thus appears to be buried.Fluorescence depolarisation measurements have been used to examine the rotational mobility of the tryptophan in the protein and in the nucleoprotein complex. In the protein alone a single rotational correlation time () of 19 ns is observed, corresponding to rotation of the entire dimeric molecule; in the native nucleoprotein complex with Pfl DNA, a of 500 ns is observed, corresponding to a rigid unit of at least 50 subunits. In neither case does the tryptophan exhibit any detectable flexibility on the subnanosecond time scale.     

Single-stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids

The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.     

Microextraction of Nuclear Proteins from Single Maize Embryos

The analysis of DNA binding proteins can be difficult when only small quantities of tissue expressing the desired protein are available. We present a protocol for the preparation of nuclear extracts from as little as 100 mg of tissue. This protocol is well suited for extraction of DNA binding proteins from tissues that are difficult to obtain in large quantities such as maize embryos.     

中华蜜蜂气味结合蛋白AcerOBP14的表达及配体结合特性分析

[目的]气味结合蛋白(odorant binding proteins,OBPs)在昆虫寄主定位、产卵地选择等行为中发挥着重要作用,明确中华蜜蜂Apis cerana cerana AcerOBP14与配体的结合特性有助于阐明中华蜜蜂嗅觉识别的分子机制.[方法]通过qRT-PCR测定OBP14在20日龄中华蜜蜂成年工蜂...     

小菜蛾触角结合蛋白PxylOBP31的鉴定与结合特性分析

【目的】触角结合蛋白(antennal binding proteins,ABPs)为昆虫气味结合蛋白(odorant binding proteins,OBPs)家族的一个亚类,是昆虫识别和响应外界环境中气味信号的载体之一,对昆虫的生存和繁衍有着重要的意义。明确触角结合蛋白在小菜蛾Plutella xylostella(L.)嗅觉识别中的作用,有助于揭示小菜蛾嗅觉识别分子机制。【方法】利用PCR技术克隆小菜蛾的一个触角结合蛋白基因;采用实时荧光定量PCR技术对该基因在小菜蛾不同发育阶段和成虫不同组织中的表达量进行分析;利用荧光竞争结合实验测试该触角结合蛋白与39种配基化合物的结合特性。【结果】成功克隆了一个小菜蛾触角结合蛋白基因,命名为Pxyl OBP31(Gen Bank登录号:KT156676)。序列分析结果显示,其开放阅读框全长411 bp,编码136个氨基酸,N端自起始位置开始21个氨基酸为信号肽,含有气味结合蛋白家族的6个保守半胱氨酸残基,预测分子量为14.74 k D,等电点为4.41。表达谱分析表明,Pxyl OBP31主要在雄蛾中表达,且交配后的雄蛾中表达量明显降低;该基因在小菜蛾触角中有较高表达,在雄蛾触角中的表达量比雌蛾触角中高近2倍。结合特性实验结果显示,Pxyl OBP31与醛、酮、萜品油烯以及邻苯二甲酸二异丁酯等物质的结合能力较强,与3种性信息素及其他烯烃与酯类结合能力弱。【结论】本研究明确了Pxyl OBP31的核苷酸序列以及发育和组织表达谱。根据qRT-PCR和荧光竞争结合实验结果,推测Pxyl OBP31蛋白可能与小菜蛾觅偶、定位寄主植物等行为有关。     

人Ha-ras基因中蛋白质特异结合DNA位点的鉴定

利用迁移率改变法和DNaseⅠ足纹法观察了Ha-ras癌基因5’端上游序列与特异结合蛋白的相互作用。用限制性内切酶XmaⅠ消化6.6kb Ha-ras基因得到约10个片段,进行3’-末端标记,与T24细胞核提取液反应,经低离子强度聚丙烯酰胺凝胶电泳,发现与蛋白质特异结合的416bp片段,位于Ha-ras基因5’-端上游1230—1646区域内,靠近转录起始点1660bp处(CAP位置)。另一个与蛋白质特异结合的389bp片段,位于转录起始点上游162—551处。     

DEK蛋白C末端DNA结合域的原核表达、纯化及其与DNA的结合活性

DEK蛋白C末端DNA结合域（简称CDB）是近年新发现的一个DEK蛋白与DNA的结合域,其中含有多个磷酸化位点,与DEK蛋白的功能密切相关。利用原核表达系统表达DEK蛋白的CDB肽段并进行纯化,具体为以pET30a（+）为载体质粒,E.coli BL21（DE3）为宿主细胞,构建重组基因工程菌,以IPTG诱导目的蛋白的表达,用NiNTA纯化的重组蛋白样品来进行SDSPAGE电泳分析,约在10.7kDa处出现明显的特征蛋白条带。凝胶迁移分析证实DEK蛋白C末端DNA结合域与DNA的结合倾向于与超螺旋型DNA相结合,同全长的DEK蛋白与DNA的结合具有类似的特点,表明DEK蛋白C末端DNA结合域在DEK蛋白与DNA的结合中可能具有一定的作用。