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71.
Experimental residual dipolar couplings (RDCs) in combination with structural models have the potential for accelerating the
protein backbone resonance assignment process because RDCs can be measured accurately and interpreted quantitatively. However,
this application has been limited due to the need for very high-resolution structural templates. Here, we introduce a new
approach to resonance assignment based on optimal agreement between the experimental and calculated RDCs from a structural
template that contains all assignable residues. To overcome the inherent computational complexity of such a global search,
we have adopted an efficient two-stage search algorithm and included connectivity data from conventional assignment experiments.
In the first stage, a list of strings of resonances (CA-links) is generated via exhaustive searches for short segments of
sequentially connected residues in a protein (local templates), and then ranked by the agreement of the experimental 13Cα chemical shifts and 15N-1H RDCs to the predicted values for each local template. In the second stage, the top CA-links for different local templates
in stage I are combinatorially connected to produce CA-links for all assignable residues. The resulting CA-links are ranked
for resonance assignment according to their measured RDCs and predicted values from a tertiary structure. Since the final
RDC ranking of CA-links includes all assignable residues and the assignment is derived from a “global minimum”, our approach
is far less reliant on the quality of experimental data and structural templates. The present approach is validated with the
assignments of several proteins, including a 42 kDa maltose binding protein (MBP) using RDCs and structural templates of varying
quality. Since backbone resonance assignment is an essential first step for most of biomolecular NMR applications and is often
a bottleneck for large systems, we expect that this new approach will improve the efficiency of the assignment process for
small and medium size proteins and will extend the size limits assignable by current methods for proteins with structural
models. 相似文献
72.
Jiajia Ma Zijia Ren Yang Ma Lu Xu Ying Zhao Chaogu Zheng Yinghui Fang Ting Xue Baolin Sun Weihua Xiao 《The Journal of biological chemistry》2009,284(50):34600-34606
73.
Yinghao Z Jun W Yuanbo C Jiachang Y Xiaohong F 《Biochemical and biophysical research communications》2005,331(1):370-374
We have attempted direct observation of the light-driven rotation of a FoF(1)-ATP motor. The FoF(1)-ATP motor was co-reconstituted by the deletion-delta subunit of FoF(1)-ATP synthase with bacteriorhodopsins (BRs) into a liposome. The BR converts radiation energy into electrochemical gradient of proton to drive the FoF(1)-ATP motor. Therefore, the light-driven rotation of FoF(1)-ATP motor has been directly observed by a fluorescence microscopy using a fluorescent actin filament connected to beta-subunit as a marker of its orientation. The rotational torque value of the Fo motor was calculated as 27.93+/-1.88pNnm. The ATP motor is expected to be a promising rotary molecular motor in the development of nanodevices. 相似文献
74.
75.
利用携带有二氢叶酸还原酶(dhfr)基因的pCI载体,实现tPA突变体(FrGGI)在CHO-dhfr^-细胞中的高效表达,获得高表达细胞株。采用分子克隆常规技术,将去除3’端非蛋白编码区的tPA突变体cDNA与pCI载体连接,构建真核表达载体pCI—tPA;采用阳离子脂质体转染法转染CHO-dhfr^-胞。经酶切及测序鉴定,证明所构建的质粒正确,转染CHO—dhfr细胞后,经过MTX加压筛选,得到了10株表达水平较高的细胞株,其活性可达每106细胞4000U/24h。以上结果为进行tPA突变体工程细胞株的筛选奠定了基础。 相似文献
76.
Identification,Design and Bio-Evaluation of Novel Hsp90 Inhibitors by Ligand-Based Virtual Screening
JianMin Jia XiaoLi Xu Fang Liu XiaoKe Guo MingYe Zhang MengChen Lu LiLi Xu JinLian Wei Jia Zhu ShengLie Zhang ShengMiao Zhang HaoPeng Sun QiDong You 《PloS one》2013,8(4)
Heat shock protein 90 (Hsp90), whose inhibitors have shown promising activity in clinical trials, is an attractive anticancer target. In this work, we first explored the significant pharmacophore features needed for Hsp90 inhibitors by generating a 3D-QSAR pharmacophore model. It was then used to virtually screen the SPECS databases, identifying 17 hits. Compound S1 and S13 exhibited the most potent inhibitory activity against Hsp90, with IC50 value 1.61±0.28 μM and 2.83±0.67 μM, respectively. Binding patterns analysis of the two compounds with Hsp90 revealed reasonable interaction modes. Further evaluation showed that the compounds exhibited good anti-proliferative effects against a series of cancer cell lines with high expression level of Hsp90. Meanwhile, S13 induced cell apoptosis in a dose-dependent manner in different cell lines. Based on the consideration of binding affinities, physicochemical properties and toxicities, 24 derivatives of S13 were designed, leading to the more promising compound S40, which deserves further optimization. 相似文献
77.
78.
苏云金芽胞杆菌营养期杀虫蛋白基因的克隆及表达分析 总被引:9,自引:0,他引:9
选择本实验室分离的苏云金芽胞杆菌李氏亚种 (subsp. Leesis) 菌株YBT833、鲇泽亚种(subsp.Aizawai) 菌株YBT-1416和库斯塔克亚种(subsp. Kurstaki)菌株YBT1535为出发菌株,以营养期杀虫蛋白基因PCR扩增的特异片段为探针,进行总DNA酶切片段的Southern杂交定位。结果显示3株菌株的营养期杀虫蛋白基因,均位于经XbaI完全消化的4~5kb大小的DNA 片段上。将该区域DNA片段回收后克隆到pUC19载体,建立了3个较基因组文库小的亚基因组文库。通过菌落原位杂交筛选和酶切鉴定分别得到3个相应的营养期杀虫蛋白基因vip83、vip14和vip15,并对其测序。DNA序列比较发现基因vip83与已知营养期杀虫蛋白基因存在5个差异碱基。将vip83、vip14基因亚克隆到苏云金芽胞杆菌大肠杆菌穿梭载体pHT315, 分别得到重组质粒pBMB8901和pBMB8902。将它们电转化到vip-的B.t.受体菌BMB171和4Q7,获得了相应的工程菌BMB8901-171,BMB8902-171,BMB8901-4Q7和BMB8902-4Q7。SDS-PAGE电泳检测均有88kD大小的蛋白表达。生物测定结果亦表明了,营养期杀虫蛋白Vip83和Vip14对鳞翅目棉铃虫、小菜蛾和甜菜夜蛾的三龄幼虫均有一定的杀虫活性;其中对小菜蛾的毒力最高,LC50值分别为28.6,31.6,45.4和37.6μL/mL。该结果为构建高效广谱工程菌提供了实际材料和理论依据。
相似文献
79.
80.
银杏叶提取物对糖尿病大鼠心肌损伤的防护作用 总被引:9,自引:0,他引:9
目的:研究银杏叶提取物(EGb)对糖尿病大鼠心肌的防护作用.方法:用光镜和透射电镜观察EGb对糖尿病大鼠心肌的形态学改变,并测定心肌组织内超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)、结构型一氧化氮合酶(cNOS)、诱导型一氧化氮合酶(iNOS)的活性及一氧化氮(NO)、丙二醛(MDA)的含量.结果:糖尿病大鼠心肌光镜下主要表现为心肌细胞空泡变性及心肌纤维局灶性溶解;电镜下主要表现为心肌线粒体肿胀,嵴变短,肌原纤维溶解;SOD活性下降,NOS、iNOS活性及MDA、NO含量增高.EGb治疗组病变明显减轻,EGb治疗组心肌组织内SOD活性明显高于糖尿病组,NOS、iNOS活性及MDA、NO含量低于糖尿病组.结论:EGb可能通过抗脂质过氧化作用和降低NO水平而对糖尿病心肌产生保护作用. 相似文献