Quantitative analysis of protein orientation in membrane environments by kinase activity

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• 作者：Wen Xiong¹ ; ² ; ^&Dagger ; ; ^{xiongwen-jan@hotmail.com" class="auth_mail" title="E-mail the corresponding author} ; Chunshan Quan¹ ; ² ; ^&Dagger ; ; ^{mikyeken@dlnu.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Xuning Zhang³ ; ^{xuningzhangchina@163.com" class="auth_mail" title="E-mail the corresponding author} ; Lina Wang⁴ ; ^{wanglina.gucas@hotmail.com" class="auth_mail" title="E-mail the corresponding author} ; Baoquan Liu¹ ; ² ; ^{lbq@dlnu.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Liming Jin¹ ; ² ; ^{jlm@dlnu.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Shengdi Fan¹ ; ² ; ^{fsd@dlnu.edu.cn" class="auth_mail" title="E-mail the corresponding author}
• 关键词：AgrC ; Protein orientation ; Liposome ; Thiol-reactive reagent ; Kinase activity
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：121
• 期：2
• 页码：242-246
• 全文大小：1502 K

文摘

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.