title: Quantitative analysis of protein orientation in membrane environments by kinase activity.
abstract: AgrC is an integral membrane receptor protein with histidine kinase activity in the accessory gene regulator (agr) quorum-sensing system of Staphylococcus aureus. In this study, proteoliposomes were used as a model to investigate AgrC orientation. Many approaches have been described to determine membrane protein orientation, but they are often complicated and time consuming. In this study, AgrC orientation in liposomes was determined by thiol-reactive reagent labeling and a kinase activity assay. Our results suggest use of a kinase activity assay could get an accurate percentage of functional protein orientation and only cost nearly one-sixth of the time compared with the method based on thiol-reactive reagent labeling. We present an effective and rapid method for determining the orientation of membrane protein kinases like AgrC.
authors.affiliation: Department of Life Science, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China; Key Laboratory of Biotechnology and Resource Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China. Electronic address: xiongwen-jan@hotmail.com.
authors.forename: Wen
authors.initials: W
authors.lastname: Xiong
authors.affiliation: Department of Life Science, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China; Key Laboratory of Biotechnology and Resource Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China. Electronic address: mikyeken@dlnu.edu.cn.
authors.forename: Chunshan
authors.initials: C
authors.lastname: Quan
authors.affiliation: School of Biological Engineering, Dalian Polytechnic University, 1 Qinggongyuan, Dalian 116034, China. Electronic address: xuningzhangchina@163.com.
authors.forename: Xuning
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhong-shan Road, Dalian 116023, China. Electronic address: wanglina.gucas@hotmail.com.
authors.forename: Lina
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Life Science, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China; Key Laboratory of Biotechnology and Resource Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China. Electronic address: lbq@dlnu.edu.cn.
authors.forename: Baoquan
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Life Science, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China; Key Laboratory of Biotechnology and Resource Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China. Electronic address: jlm@dlnu.edu.cn.
authors.forename: Liming
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Life Science, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China; Key Laboratory of Biotechnology and Resource Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China. Electronic address: fsd@dlnu.edu.cn.
authors.forename: Shengdi
authors.initials: S
authors.lastname: Fan
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D055162:Biocatalysis; D057075:Enzyme Assays; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D053038:Quorum Sensing; D013211:Staphylococcus aureus / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011510:Proteolipids; D013438:Sulfhydryl Compounds; C020485:proteoliposomes; D011494:Protein Kinases; C531166:AgrC protein, Staphylococcus
keywords: AgrC; Kinase activity; Liposome; Protein orientation; Thiol-reactive reagent