结核杆菌小分子热休克蛋白Hsp16.3的高效自发再折叠和再组装

来自结核杆菌的小分子热休克蛋白Hsp16.3以九聚体的形式存在.用三种不同的强变性条件（100℃加热15 min,12 mol/L脲或8 mol/L盐酸胍处理4 h）将Hsp16.3变性, 然后通过冷却或透析使之复性,并利用孔径梯度聚丙烯酰胺凝胶电泳和圆二色性光谱比较了变性-复性前后Hsp16.3的各个层次高级结构.结果显示,变性的Hsp16.3几乎可以完全恢复至天然构象,这表明小分子热休克蛋白Hsp16.3具有很强的自发折叠和组装能力.     

结核杆菌小分子热休克蛋白Hsp16.3的高效自发再折叠和再组装

来自结核杆菌的小分子热休克蛋白Hsp16.3以九聚体的形式存在.用三种不同的强变性条件(100℃加热15 min,12 mol/L脲或8 mol/L盐酸胍处理4 h)将Hsp16.3变性, 然后通过冷却或透析使之复性,并利用孔径梯度聚丙烯酰胺凝胶电泳和圆二色性光谱比较了变性-复性前后Hsp16.3的各个层次高级结构.结果显示,变性的Hsp16.3几乎可以完全恢复至天然构象,这表明小分子热休克蛋白Hsp16.3具有很强的自发折叠和组装能力.     

拟南芥DG1参与叶绿体早期光合蛋白合成功能

拟南芥中已有466个PPR蛋白,已有研究证实许多PPR蛋白参与细胞器基因表达的转录后调节,但大部分PPR蛋白分子作用机制尚不清楚.Delayed greening 1(DG1)是定位于叶绿体中的的PPR蛋白,研究结果证实该蛋白是通过与SIG6因子相互作用降低PEP转录活性从而影响叶绿体早期发育.本研究利用拟南芥Dg1基因功能缺陷型突变体研究了DG1蛋白对光系统蛋白复合体组成及其光转化效率的影响.77K荧光发射光谱分析发现dg1突变体幼叶PSII中电子传递速度明显低于野生型,而成熟叶片与野生型基本一致;蓝绿温和胶分析结果表明:相对于野生型在dg1突变体新生叶中PSII、PS玉及其超聚复合物含量均有不同程度降低;进一步温和胶二向电泳及蛋白免疫印迹分析显示,在dg1突变体新生叶中,由叶绿体编码的光系统蛋白复合物组成亚基含量显著降低,而核编码复合物组成亚基含量与野生型相比没有明显区别.上述实验结果进一步确定了DG1蛋白是通过调控叶绿体编码基因的表达进而调节光系统复合物的生物合成与组装,最终影响拟南芥叶绿体早期发育.因此,我们认为DG1蛋白对于叶绿体发育早期光合蛋白的合成是必需的.     

位于大鼠肌乳酸脱氢酶(LDHA)N端结构中的点突变对其四体稳定性的影响

乳酸脱氢酶(LDH)为典型的相同亚基的四聚体蛋白,其N端结构十分特殊,包含着一个α螺旋和一个β结构,它们对于LDH四级结构的形成起着十分重要的作用.为了进一步研究N端结构中的特殊残基对其四体形成的影响,构建了Leu3Pro(L3P)和Ile8Glu(I8E)两个突变体.在同野生型LDH(WT)稳定性实验的比较中发现,两个突变体的四级结构稳定性均有不同程度的降低：野生型酶在0.6~0.8 mol/L GdmCl中解离为二体;L3P在天然态时虽是四体的形式,但在0.3 mol/L GdmCl中已解离成二体;而I8E在天然态时主要以二体的形式存在,并且其KM升高,k_cat降低,且可以被Na₂SO₄激活.上述结果充分证明了N端结构,特别是在β结构中引入一个负电荷,能够显著影响亚基的相互作用,影响四级结构的稳定性.     

血红素对大肠杆菌铁蛋白稳定性及自组装的影响

大肠杆菌铁蛋白(bacterioferritin,BFR)是由同源亚基24聚体组成的高度对称的八面体壳状结构,其中处在C2对称轴的每两个亚基之间即存在一个血红素分子。为了探讨血红素对细菌铁蛋白结构稳定性及在蛋白质自组装过程中的影响,通过定点突变将第52位蛋氨酸突变为丙氨酸以去除血红素分子,并应用体积排阻色谱、透射电镜、紫外-可见光吸收光谱及圆二色谱等手段对突变蛋白M52A的聚合态、二级结构及热力学性质等进行分析。结果表明:去除血红素虽然对铁蛋白的自组装没有明显影响,但是对其热稳定性产生较大影响,突变后蛋白质的T_m值为54.3℃,远小于野生型铁蛋白T_m值69.9℃,且经过氯化血红素与同浓度下该突变体M52A化学诱导结合后,发现其T_m值重新恢复至67.7℃。由此可知,血红素对大肠杆菌铁蛋白稳定性影响很大,且具有调控铁蛋白稳定性的作用。     

Hsp70蛋白自身磷酸化对其分子伴侣功能的影响

近年对分子伴侣蛋白Hsp70作用机制的研究发现,其ATP功能区域X光晶体结构有一个新的钙离子结合区域,这个新的功能区域与Hsp70分子的ADP结合、ATP水解及合成有关.有报道认为Hsp70蛋白的NDP激酶样作用,通过形成酸不稳定性自身磷酸化中间体催化γ 磷酸基团在ATP和ADP间传递,组氨酸H89与这个新的区域有密切关系,有可能与Hsp70蛋白形成自身磷酸化中间体有关.本研究运用基因定位诱导突变技术,将89位组氨酸以丝氨酸替代(H89S),通过比较Hsp70野生型及突变型蛋白的自身磷酸化过程的改变,及其对Hsp70蛋白体外荧光素酶活性影响的不同,初步探讨Hsp70作用机制.结果发现,突变的H89S蛋白自身磷酸化过程及体外变性荧光素酶重折叠受到抑制.野生型蛋白未受到影响,野生型Hsp70可以形成酸不稳定的自身磷酸化中间体,产生CDP依赖性解磷酸反应,而H89S突变型蛋白不能形成这种反应.89位组氨酸点突变能显著降低ATP酶交换反应及体外变性荧光素酶重折叠水平,但它的自身磷酸化可能并非唯一必需的介导位点或只是一个选择性的功能侧链.     

人副流感病毒3型HN蛋白十一肽重复序列保守氨基酸突变分析

为确定人副流感病毒3型(Human parainfluenza virus type 3,hPIV3)病毒包膜表面血凝素神经氨酸酶(Hemagglutinin-neuraminidase,HN)糖蛋白茎部区十一肽重复序列中保守氨基酸中具有关键性作用的位点,进一步探讨HN蛋白茎部区在融合机制中的重要作用。结合定点突变和同源重组技术将HN蛋白茎部区十一肽重复序列中5个保守氨基酸位点(I102、P111、L114、S119、I125)突变为丙氨酸(Alanine,A),通过痘苗病毒-T7聚合酶系统在BHK-21细胞中表达突变蛋白,定性定量检测各突变体蛋白的促细胞融合活性、受体结合活性、神经氨酸酶活性和半融合活性。突变体蛋白I102A、P111A、L114A、S119A、I125A的促细胞融合活性均有不同程度下降,依次为野生型的6%、16%、14%、87%和4%,除S119A外其余4个突变型与野生型相比差别均具有统计学意义(P0.01);突变体蛋白I102A、P111A、L114A、S119A、I125A的受体结合活性也出现不同程度下降,依次分别为野生型的32.2%、77.4%、74.2%、83.9%和38.7%,其中I102A和I125A的受体结合活性与野生型相比差别具有统计学意义(P0.01);突变体蛋白I102A、P111A、L114A、S119A、I125A的神经氨酸酶活性分别为野生型的66.5%、73.1%、69.1%、76.1%和72.8%,与野生型相比差别无统计学意义(P0.05)。结果表明:茎部区十一肽重复序列对hPIV3HN蛋白的促细胞融合活性和受体结合活性具有重要意义。该区域氨基酸I102、P111、L114、I125具有关键作用,推测其能通过影响头部区受体结合活性或是与融合蛋白的相互作用等不同方式导致HN蛋白结构功能发生改变。     

GST pull-down验证流感病毒PB1-F2蛋白与热休克蛋白40的相互作用

为体外验证流感病毒PB1-F2与热休克蛋白Hsp40相互作用,通过两个方向的GST pull-down试验验证PB1-F2与Hsp40的相互作用。构建GST-多肽融合蛋白原核表达载体pGEX-6P-1-PB1-F2和pGEX-6P-1-Hsp40,并在大肠杆菌(E.co-li)BL21中诱导表达;构建真核表达载体pLEGFP-Hsp40及pCAGGS-PB1-F2,并分别转染293T细胞使其表达Hsp40及PB1-F2融合蛋白,然后进行GST pull-down试验验证二者的相互作用。成功地构建了两种蛋白的各种表达载体,经表达、纯化获得了可溶性的GST-多肽融合蛋白,GST pull-down试验正反两方向都证实了PB1-F2与Hsp40的相互作用,初步证实了流感病毒PB1-F2在体外能与Hsp40发生相互作用。     

GST-pulldown体外研究LRP16与NF-KB/p65相互作用的功能表位

目的:构建核转录因子NF-κB/p65亚基的功能表位载体,采用GST-pulldown探究p65与LRP16体外相互作用的功能表位.方法:以真核表达质粒pcDNA3.1-p65为模板,PCR扩增一组p65功能表位的基因片段,经测序后,克隆至pcDNA3-flag载体上,用BamH Ⅰ/Xho Ⅰ双酶切重组质粒鉴定目的片段大小,然后采用GST-pulldown实验验证LRP16与p65之间的相互作用.结果:构建了p65的5个功能表位的表达载体且可用于体外转录翻译,GST-pulldown体外研究表明,p65蛋白N端RHD结构域是与LRP16相互作用所必需的,p65的RHD结构域的缺失完全消除了p65与LRP16的体外相互作用.结论:GST-pulldown实验验证了LRP16与p65存在体外直接相互作用,并具体分析了LRP16与p65相互作用的表位.     

重组人CK2β亚基的原核表达、纯化与鉴定

将构建成功的人蛋白激酶CK2β亚基cDNA的重组质粒, 转化大肠杆菌BL21 (DE3), 在IPTG诱导下表达. 表达蛋白大多数以不溶形式存在. 6L(约10.2 g)表达菌抽提得到约20 mg的可溶性表达产物, 通过P11磷酸纤维素一步层析分离, 得到6.8 mg纯化蛋白. SDS-聚丙烯酰胺凝胶电泳结果显示纯化的蛋白为一分子质量26 ku的单一蛋白带.蛋白质印迹结果证明：纯化的表达产物与抗人CK2β抗体可发生特异性免疫反应. CK2β亚基对CK2α有激活作用, 纯化的CK2α和β亚基在等摩尔混合时即可组成有最大生物活性的全酶. 实验结果有力地证明了克隆表达与纯化的重组蛋白是人蛋白激酶CK2β亚基.     

Inter-subunit Cross-linking Suppressed the Dynamic Oligomeric Dissociation of Mycobacterium tuberculosis Hsp16.3 and Reduced Its Chaperone Activity

Small heat shock proteins (sHsps) usually exist as dynamic oligomers and oligomeric dissociation was believed to be a prerequisite for their chaperone activities. The truth of this hypothesis was verified in our present study on Hsp16.3, one member of sHsps from Mycobacterium tuberculosis, mainly by utilizing chemical cross-linking. Analysis using size exclusion chromatography demonstrated that the heat-induced oligomeric dissociation of Hsp16.3 was severely blocked due to highly efficient inter-subunit cross-linkages generated by chemical cross-linking, as well as its chaperone activity being reduced. Further analysis by non-denaturing pore gradient polyacrylamide gel electrophoresis and fluorescence spectrometry revealed that the dynamic oligomeric dissociation/reassociation process of Hsp16.3 at room temperature was suppressed by inter-subunit cross-linkages, accompanied by significantly decreased exposure of hydrophobic surfaces that are usually hidden in oligomers. These findings supported the hypothesis that substrate-binding sites of sHsps are exposed presumably by dissociation of larger oligomers into smaller active oligomers, and therefore such a dissociation process could be adjusted to modulate chaperone activities.     

Chaperone-Like Activity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Hsp16.3 Does Not Require Its Intact (Native) Structures

Small heat shock proteins (sHsps) were found to exhibit efficient chaperone-like activities under stress conditions although their native structures are severely disturbed. Here, using an alternative approach (site-directed mutagenesis), we obtained two structurally and functionally distinct Mycobacterium tuberculosis Hsp16.3 single-site mutant proteins. The G59W mutant protein (with Gly59 substituted by Trp) is capable of exhibiting efficient chaperone-like activity even under non-stress conditions although its secondary, tertiary, and quaternary structures are very different from that of the wild type protein. By contrast, the G59A mutant protein (with Gly59 substituted by Ala) resembles with the wild type protein in structure and function. These observations suggest that the Gly59 of the Hsp16.3 protein is critical for its folding and assembly. In particular, we propose that the exhibition of chaperone-like activity for Hsp16.3 does not require its intact (native) structures but requires the disturbance of its native structures (i.e., the native structure-disturbed Hsp16.3 retains its chaperone-like activity or even becomes more active). In addition, the behavior of such an active mutant protein (G59W) also strongly supports our previous suggestion that Hsp16.3 exhibits chaperone-like activity via oligomeric dissociation.     

甲基对硫磷降解菌假单胞菌WBC-3的筛选及其降解性能的研究

从农药污染土样中分离出的一株细菌具有彻底降解甲基对硫磷的能力。该菌经生理生化特性分析和16S rDNA序列同源性分析,鉴定为假单胞菌属,命名为Pseudomonas sp.WBC\|3。该菌在pH7～8、温度23℃～30℃范围内均生长良好,对甲基对硫磷的耐受浓度在单纯无机盐培养基中可达到800mg/L,在含有01%葡萄糖的培养基中可达到2000mg/L。该菌能够以甲基对硫磷作为唯一碳源、氮源,将其彻底降解作为生长基质,对于300mg/L甲基对硫磷的降解速度达15mg/L\5h,于22h后达到其稳定生长期。该菌对于多种有机磷农药及部分芳烃类化合物具有生化代谢能力。从该菌的细胞周质组分中纯化出的有机磷水解酶在SDSPAGE胶上显示为分子量约为33.5×103的条带。     

Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.     

人肝脏组织亚细胞组分中含硒蛋白的分离与测定

硒是生物必需的微量元素 ,具有重要的生物化学功能 ,与人类和动物的健康及疾病密切相关[1,2 ] .生物体内 80 %以上的硒是以与蛋白质结合的形式存在 ,所以目前大量的研究集中在硒蛋白的分离、鉴定和功能研究等方面[3 ] .从 1998年起 ,我们对正常人肝组织中硒和含硒蛋白的亚细胞分布作了一些探索性的工作 ,发现含硒蛋白在各亚细胞组分中的分布有显著的不同[4 ,5] .前期工作采用的凝胶过滤柱层析法研究含硒蛋白 ,该法分离量大 ,但分辨率较差 ,分子量相近的蛋白质难以区分[4 ,5] .本工作采用ＳＤＳ 聚丙烯酰胺凝胶电泳 (ＳＤＳ ＰＡＧＥ)分离人…     

Probing the roles of the only universally conserved leucine residue (Leu122) in the oligomerization and chaperone-like activity of Mycobacterium tuberculosis small heat shock protein Hsp16.3

To understand the role of the only universally conserved hydrophobic residue among all the members of the sHsp family, this extremely well conserved Leu122 residue in Hsp16.3 was replaced by valine, alanine, asparigine, or aspartate residues. Only very small amounts of the L122D and L122N mutant Hsp16.3 proteins were expressed in the transformed E. coli; however, both the L122V and L122A were readily expressed. The L122V and L122A mutant proteins had similar oligomeric structures to the wild-type protein at room temperature. Examination of the L122A mutant protein by native pore gradient PAGE and CD spectroscopy, however, revealed a smaller oligomeric size and different secondary structure at 37°C. Both L122V and L122A mutant proteins exhibited significantly lowered chaperone activities. Observations reported here suggest a very important role of this only universally conserved Leu residue in both the formation of specific oligomeric structures and the molecular chaperone activities of Hsp16.3.     

The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress

The nuclear-encoded chloroplast-localized Hsp21 is an oligomeric heat shock protein (Hsp), belonging to the protein family of small Hsps and alpha-crystallins. We have investigated the effects of high temperature and oxidation treatments on the structural properties of Hsp21, both in purified recombinant form and in transgenic Arabidopsis thaliana plants engineered to constitutively overexpress Hsp21. A conformational change was observed for the 300 kDa oligomeric Hsp21 protein during moderate heat stress (< or =40 degrees C) of Arabidopsis plants, as judged by a shift to lower mobility in non-denaturing electrophoresis. Similar changes in mobility were observed when purified recombinant Hsp21 protein was subjected to an oxidant. Exposure of Hsp21 protein to temperatures above 70 degrees C led to irreversible aggregation, which was prevented in presence of the reductant dithiothreitol. The transgenic plants that constitutively overexpressed Hsp21 were more resistant to heat stress than were wildtype plants when the heat stress was imposed under high light conditions. These results suggest that the physiological role of Hsp21 involves a response to temperature-dependent oxidative stress.     

Stepwise disassembly and apparent nonstepwise reassembly for the oligomeric RbsD protein

Many cellular proteins exist as homo-oligomers. The mechanism of the assembly process of such proteins is still poorly understood. We have previously observed that Hsp16.3, a protein exhibiting chaperone-like activity, undergoes stepwise disassembly and nonstepwise reassembly. Here, the disassembly and reassembly of a nonchaperone protein RbsD, from Escherichia coli, was studied in vitro. The protein was found to mainly exist as decamers with a small portion of apparently larger oligomeric forms, both of which are able to refold/reassemble effectively in a spontaneous way after being completely unfolded. Disassembly RbsD intermediates including pentamers, tetramers, trimers, dimers, and monomers were detected by using urea-containing pore gradient polyacrylamide gel electrophoresis, while only pentamers were detected for its reassembly. The observation of stepwise disassembly and apparent nonstepwise reassembly for both a chaperone protein (Hsp16.3) and a nonchaperone protein (RbsD) strongly suggests that such a feature is most likely general for homo-oligomeric proteins.