title: Functional reconstitution of Staphylococcus aureus truncated AgrC histidine kinase in a model membrane system.
abstract: The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrCTM5-6C and AgrCTM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrCTM5-6C and AgrCTM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrCTM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrCTM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrCTM5-6C in detergent micelles. The reconstitution of AgrCTM5-6C or AgrCTM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system.
authors.affiliation: Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Wang
authors.forename: Chunshan
authors.initials: C
authors.lastname: Quan
authors.forename: Baoquan
authors.initials: B
authors.lastname: Liu
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Wang
authors.forename: Wen
authors.initials: W
authors.lastname: Xiong
authors.forename: Pengchao
authors.initials: P
authors.lastname: Zhao
authors.forename: Shengdi
authors.initials: S
authors.lastname: Fan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D004789:Enzyme Activation; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008567:Membranes, Artificial; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D011494:Protein Kinases; C531166:AgrC protein, Staphylococcus