不同电性脂质体对金黄色葡萄球菌组氨酸激酶AgrC跨膜镶嵌效率的影响

doi:10.7503/cjcu20140057

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (9): 1901.doi: 10.7503/cjcu20140057

不同电性脂质体对金黄色葡萄球菌组氨酸激酶AgrC跨膜镶嵌效率的影响

熊文^1,², 权春善^1,²(), 王丽娜³, 张旭宁⁴, 赵晶^1,², 郑维^1,², 范圣第^1,²

1. 大连民族学院生物技术与资源利用国家民委-教育部重点实验室大连 116600
2. 大连民族学院生物技术与资源利用国家民委生命科学学院, 大连 116600
3. 中国科学院大连化学物理研究所, 大连 116023
4. 大连工业大学生物工程学院, 大连 116034

收稿日期:2014-01-17 出版日期:2014-09-10 发布日期:2019-08-01
作者简介:联系人简介: 权春善, 女, 博士, 教授, 主要从事化学生物学研究. E-mail: mikyeken@dlnu.edu.cn。
基金资助:
国家自然科学基金(批准号: 21272031)和大连民族学院研究生创新基金项目(批准号: YCX2013016)资助

Effect of Different Charged Liposomes on Transmembrane Incorporation Efficiency of the Staphylococcus Aureus Receptor Histidine Kinase AgrC^†

XIONG Wen^1,², QUAN Chunshan^1,^2,^*(), WANG Lina³, ZHANG Xuning⁴, ZHAO Jing^1,², ZHENG Wei^1,², FAN Shengdi^1,²

1. Key Laboratory of Biotechnology and Resource Utilization, State Ethnic Affairs Commission and Ministry of Education Dalian 116600, China
2. Department of Life Science, Dalian Nationalities University, Dalian 116600, China
3. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
4. School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China

Received:2014-01-17 Online:2014-09-10 Published:2019-08-01
Contact: QUAN Chunshan E-mail:mikyeken@dlnu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(No.21272031) and the Graduate Student Innovation Foundation of Dalian Nationalities University, China(No.YCX2013016)

摘要/Abstract

摘要：

通过改变脂质体中磷脂成分, 构建了不同电性的脂质体. 利用表面活性剂介导方法, 将截短的金黄色葡萄球菌细胞膜上的组氨酸激酶AgrC(AgrC_TM6-7C)蛋白重构到不同电性的脂质体上. 结果表明, 阴离子脂质体对AgrC_TM6-7C蛋白的镶嵌效率明显高于阳离子脂质体, 约60%~70%镶嵌至阴离子脂质体中的AgrC_TM6-7C蛋白的细胞质域朝向脂质体囊泡的外部, 并保持较高活性. 利用圆二色光谱比较了AgrC_TM6-7C蛋白在表面活性剂胶束和脂质体中的二级结构稳定性, 发现阴离子脂质体对AgrC_TM6-7C蛋白的二级结构具有一定的保护作用, 可明显提高蛋白的热稳定性.

关键词: 金黄色葡萄球菌, 组氨酸激酶AgrC, 脂质体, 蛋白脂质体, 膜蛋白

Abstract:

AgrC is a membrane-embedded histidine kinase in Staphylococcus aureus that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm so as to regulate and control a series of related pathogenic gene expression. However, for the complexity of cell membrane, it turns to be difficult to study AgrC on bacterial cell membrane directly. Many researches tried to take advantage of proteoliposome which could provide an approximate natural membrane environment and keep the protein activity to study the structure and function of membrane proteins. In order to build a transmembrane protein incorporation system with a relatively high incorporation efficiency, function activity and stability, various factors were considered, such as the impact of different charge polarity head on protein transmembrane incorporation efficiency. Here, four kinds of different charged liposomes were prepared by changing the phospholipid components in liposome and a detergent-mediated method was used to reconstitute truncated AgrC protein(AgrC_TM6-7C) into them. The results showed that negatively charged liposomes leaded to a higher incorporation efficiency compared with the positively charged one and about 60%—70% C-terminal cytoplasmic domain of AgrC_TM6-7C protein reconstituted into negatively charged liposomes were outside-orientated which remained a highly kinase activity. Furthermore, from circular dichroism detection result, we can draw a conclusion that liposome has certain protective effect on AgrC_TM6-7Cprotein secondary structure and improve the thermal stability of the protein.

Key words: Staphylococcus aureus, AgrC, Liposome, Proteoliposome, Membrane protein

中图分类号:

TrendMD:

熊文, 权春善, 王丽娜, 张旭宁, 赵晶, 郑维, 范圣第. 不同电性脂质体对金黄色葡萄球菌组氨酸激酶AgrC跨膜镶嵌效率的影响. 高等学校化学学报, 2014, 35(9): 1901.

XIONG Wen, QUAN Chunshan, WANG Lina, ZHANG Xuning, ZHAO Jing, ZHENG Wei, FAN Shengdi. Effect of Different Charged Liposomes on Transmembrane Incorporation Efficiency of the Staphylococcus Aureus Receptor Histidine Kinase AgrC^†. Chem. J. Chinese Universities, 2014, 35(9): 1901.

图/表 7

Table 1 Properties of different liposomes formed with different charged phospholipids

Lipid composition	DOTAP-DPPC	DOTAP-DOPC	DPPA-DPPC	DPPA-DOPC
Particle size/nm	357	200	184	128
PI	0.399	0.331	0.408	0.339
Zeta potential/mV	72.7	80.1	-79.7	-75.0

Fig.1 TEM images of liposomes (A) DOTAP-DPPC; (B) DOTAP-DOPC; (C) DPPA-DPPC; (D) DPPA-DOPC.

Fig.2 Fluorescence intensity of AgrCTM6-7C-GFP in different charged liposomes before(■) and after(■) centrifugation The percentages represent incorporation efficiency respectively. a. DPPA-DPPC; b. DPPA-DOPC; c. DOTAP-DPPC; d. DOTAP-DOPC.

Fig.3 Fluorescence microscopy images of reconstituted proteoliposomes (A) DPPA-DPPC; (B) DPPA-DOPC; (C) DOTAP-DPPC; (D) DOTAP-DOPC.

Fig.4 Kinase activity of AgrCTM6-7C proteoliposome

Fig.5 Circular dichroism spectra of secondary structure changes of AgrCTM6-7C in brij-35 micelles(a) and in liposomes(b) along with a temperature gradient

Fig.6 Fluorescence images of 5-IAF-labelled AgrCTM6-7C proteoliposomes Tube 1: proteoliposomes were solubilized with detergents and labelled with 5-IAF; tube 2: proteoliposomes were treated with AmdiS, solubilized with detergents and labelled with 5-IAF; tube 3: proteoliposomes were labelled with 5-IAF; tube 4: proteoliposomes were treated with NEM and labelled with 5-IAF.

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不同电性脂质体对金黄色葡萄球菌组氨酸激酶AgrC跨膜镶嵌效率的影响

Effect of Different Charged Liposomes on Transmembrane Incorporation Efficiency of the Staphylococcus Aureus Receptor Histidine Kinase AgrC^†

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不同电性脂质体对金黄色葡萄球菌组氨酸激酶AgrC跨膜镶嵌效率的影响

Effect of Different Charged Liposomes on Transmembrane Incorporation Efficiency of the Staphylococcus Aureus Receptor Histidine Kinase AgrC†

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Effect of Different Charged Liposomes on Transmembrane Incorporation Efficiency of the Staphylococcus Aureus Receptor Histidine Kinase AgrC^†