A genetic approach to analyzing membrane protein topology

Fusions of the secreted protein alkaline phosphatase to an integral cytoplasmic membrane protein of Escherichia coli showed different activities depending on where in the membrane protein the alkaline phosphatase was fused. Fusions to positions in or near the periplasmic domain led to high alkaline phosphatase activity, whereas those to positions in the cytoplasmic domain gave low activity. Analysis of alkaline phosphatase fusions to membrane proteins of unknown structure may thus be generally useful in determining their membrane topologies.     

Bacterial motility: membrane topology of the Escherichia coli MotB protein

The MotB protein of Escherichia coli is an essential component of the force generators that couple proton movement across the cytoplasmic membrane to rotation of the flagellar motors. The membrane topology of MotB was examined to explore the possibility that it might form a proton channel. MotB--alkaline phosphatase fusion proteins were constructed to identify likely periplasmic domains of the MotB molecule. Fusions distal to a putative membrane-spanning segment near the amino terminus of MotB exhibited alkaline phosphatase activity, indicating that an extensive carboxyl-terminal portion of MotB may be located on the periplasmic side of the membrane. Protease treatment of MotB in spheroplasts confirmed this view. The simple transmembrane organization of MotB is difficult to reconcile with a role as a proton conductor.     

拟南芥IAA7的原核表达及稳定性分析

以p GEX–KG为基本骨架,构建了拟南芥IAA7蛋白的原核表达载体,用IPTG诱导IAA7在3种大肠杆菌表达菌株Rosetta、BL21和Tuner中表达,利用GST SefiroseTM resin亲和树脂分离纯化GST–IAA7融合蛋白,并分析重组蛋白在体外保存时的稳定性。结果表明:GST–IAA7融合蛋白在Rosetta菌株中于25℃和0.4 mmol/L IPTG诱导下表达较好;蛋白酶抑制剂苯甲基磺酰氟(PMSF)可显著延长GST–IAA7在体外保存的时间,最后利用凝血酶切除GST标签后获得了纯化的IAA7蛋白质。     

具有分布时滞的双向联想记忆脉冲神经网络的全局指数稳定性

讨论了一类具有Cohen-Grossberg动力和分布时滞的脉冲型双向联想记忆神经网络的全局指数稳定性.利用矩阵理论和同伦映射,构造了恰当的李亚普诺夫泛函,获得了这种类型神经网络平衡点的存在性和唯一性定理,并得到了这种平衡点为全局指数稳定的充分条件.所获结论推广了Hopfield、BAM和Cohen-Grossberg等神经网络模型的相关结论.     

酵母甘露糖蛋白对白葡萄酒酒石和蛋白质稳定性的影响

研究了添加一定量甘露糖蛋白对干白葡萄酒酒石、蛋白质稳定性的影响。结果表明,甘露糖蛋白对白葡萄酒蛋白质稳定性有较好的作用,可以降低皂土的用量,但不能取代皂土的作用。添加甘露糖蛋白后,干白葡萄酒的初始电导率值增加;用极点电导率测试仪检测干白葡萄酒的酒石稳定性,电导率下降值减小,酒石稳定性提高。说明添加甘露糖蛋白可以解决干白葡萄酒连续冷稳处理不易解决的酒石酸钙稳定性问题。     

Herpesviral protein networks and their interaction with the human proteome

The comprehensive yeast two-hybrid analysis of intraviral protein interactions in two members of the herpesvirus family, Kaposi sarcoma-associated herpesvirus (KSHV) and varicella-zoster virus (VZV), revealed 123 and 173 interactions, respectively. Viral protein interaction networks resemble single, highly coupled modules, whereas cellular networks are organized in separate functional submodules. Predicted and experimentally verified interactions between KSHV and human proteins were used to connect the viral interactome into a prototypical human interactome and to simulate infection. The analysis of the combined system showed that the viral network adopts cellular network features and that protein networks of herpesviruses and possibly other intracellular pathogens have distinguishing topologies.     

重组猪抑制素蛋白质中毒素残留检测及其理化稳定性

为探索纯化的重组猪抑制素蛋白质毒素残留情况以及重组猪抑制素蛋白质稳定性,本研究利用鲎试剂、气相色谱和SDS-PAGE方法对重组猪抑制素蛋白质中的细菌内毒素、β-巯基乙醇的残留量及其蛋白质在不同温度、极端p H值和反复冻融条件下的稳定性进行检测。结果显示,经过3次等电点沉淀洗涤后,重组猪抑制素蛋白质中细菌内毒素和β-巯基乙醇的残留量大幅减少。通过在不同温度下的处理发现,重组猪抑制素蛋白质在4℃环境下较稳定,可以长期保存;但在37℃时,稳定性快速下降,随着温度上升而降解加速。通过极端p H值和反复冻融试验发现,重组猪抑制素蛋白质对极端p H值和反复冻融具有较好的耐受性。     

冬小麦谷粒产量,谷粒蛋白产量,谷粒蛋白含量和稳定性分析

基因型与环境的互作对基因型筛选具有重要作用,并且使品种筛选变得复杂.本文对法国27个冬小麦品种在27个生长环境中获得的试验数据进行了分析比较,同时对高谷粒产量(GY),谷粒蛋白产量(GPY),谷粒蛋白含量(GPC)和稳定性的几种方法进行了筛选:Kang秩和方法(指标1:相同权重的3种谷粒性状和Shukla稳定性方差δ2)以及5种衍生的秩和指标(指标2～6:3种谷粒性状高于δ2 2～6倍的权重),稳定性方差(s2)和回归系数(b),研究它们之间的秩相关性以及它们与3种谷粒性状的相关性.结果表明:3种稳定性统计δ2,s2和b对同时筛选3种谷粒性状指标和稳定性都是非常有用的方法.对于GPY的筛选,指标1秩和方法比稳定性方差s2略保守.指标2比指标1和稳定方差s2更好地筛选到高GY和高GPY.指标3,指标4,指标5和指标6更适合于初始筛选3种谷粒性状.所有的稳定性统计方法都能很好与3种谷粒性状秩相关.稳定性方差s2和回归系数b之间在除了GY之外的3种谷粒性状中秩相关性不高,稳定性方差δ2和稳定性方差s2之间在3种谷粒性状中都具有很强的秩相关性.3种稳定性统计在高产和低产的环境中重复性不高,同样,在2个年份之间的重复性也可以忽略.但是,在随机抽取的4个环境之间的重复性却非常高.     

Influence of interior packing and hydrophobicity on the stability of a protein

Protein interiors contain many tightly packed apolar atoms in a nearly crystalline state. Both shielding of apolar atoms from solvent and efficient interior packing arrangements affect protein stability, but their relative importance is unclear. To separate these effects, the stabilities of wild-type and mutant gene V proteins from bacteriophage fl were studied by measuring resistance to denaturation. The effects of subtle interior packing changes, both separate from and combined with changes in buried side chain hydrophobicity, were measured. For the interior apolar-to-apolar substitutions studied, the two effects were of the same magnitude and alteration of packing without accompanying hydrophobicity changes substantially destabilized the protein.     

Relating three-dimensional structures to protein networks provides evolutionary insights

Most studies of protein networks operate on a high level of abstraction, neglecting structural and chemical aspects of each interaction. Here, we characterize interactions by using atomic-resolution information from three-dimensional protein structures. We find that some previously recognized relationships between network topology and genomic features (e.g., hubs tending to be essential proteins) are actually more reflective of a structural quantity, the number of distinct binding interfaces. Subdividing hubs with respect to this quantity provides insight into their evolutionary rate and indicates that additional mechanisms of network growth are active in evolution (beyond effective preferential attachment through gene duplication).     

Antagonistic control of disease resistance protein stability in the plant immune system

Pathogen recognition by the plant immune system is governed by structurally related, polymorphic products of disease resistance (R) genes. RAR1 and/or SGT1b mediate the function of many R proteins. RAR1 controls preactivation R protein accumulation by an unknown mechanism. We demonstrate that Arabidopsis SGT1b has two distinct, genetically separable functions in the plant immune system: SGT1b antagonizes RAR1 to negatively regulate R protein accumulation before infection, and SGT1b has a RAR1-independent function that regulates programmed cell death during infection. The balanced activities of RAR1 and SGT1, in concert with cytosolic HSP90, modulate preactivation R protein accumulation and signaling competence.     

ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.     

Identification of SCF ubiquitin ligase substrates by global protein stability profiling

Ubiquitin-mediated proteolysis regulates all aspects of cellular function, and defects in this process are associated with human diseases. The limited number of identified ubiquitin ligase-substrate pairs is a major bottleneck in the ubiquitin field. We established and applied genetic technologies that combine global protein stability (GPS) profiling and genetic perturbation of E3 activity to screen for substrates of the Skp1-cullin-F-box (SCF) ubiquitin ligase in mammalian cells. Among the >350 potential substrates identified, we found most known SCF targets and many previously unknown substrates involved in cell cycle, apoptosis, and signaling pathways. Exploring cell cycle-stage stability, we found that several substrates used the SCF and other E3s in different cell cycle stages. Our results demonstrate the potential of these technologies as general platforms for the global discovery of E3-substrate regulatory networks.     

岩原鲤三种同工酶的组织特异性研究

采用聚丙烯酰胺凝胶垂直板电泳,对岩原鲤(Procypris rabaudi)6种组织(眼、脑、心、肝、肾、肌肉)的乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、酯酶(EST)3种同工酶进行了初步研究,并对其位点及酶谱表型进行了分析.结果表明,岩原鲤3种同工酶系统具有不同程度的组织特异性.LDH检测到由3个基因位点编码,Ldh-C位点仅在肝脏组织中表达:s-MDH在6种组织中均有表达,其中在肝脏和肾脏中的活性表达较强,m-MDH在脑、心脏、肾脏和肌肉4种组织中均有表达;EST检测到由5个基因位点编码,其同工酶酶谱复杂,Est-2为6种组织的共有谱带,在肝脏中的活性表达较强.     

Rheology and microscopic topology of entangled polymeric liquids

The viscoelastic properties of high molecular weight polymeric liquids are dominated by topological constraints on a molecular scale. In a manner similar to that of entangled ropes, polymer chains can slide past but not through each other. Tube models of polymer dynamics and rheology are based on the idea that entanglements confine a chain to small fluctuations around a primitive path that follows the coarse-grained chain contour. Here we provide a microscopic foundation for these highly successful phenomenological models. We analyze the topological state of polymeric liquids in terms of primitive paths and obtain parameter-free, quantitative predictions for the plateau modulus, which agree with experiment for all major classes of synthetic polymers.