Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System

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 AgrC(TM5-6C) and AgrC(TM5-6C)-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrC(TM5-6C) and AgrC(TM5-6C)-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 AgrC(TM5-6C) was almost exclusively oriented towards the inside of the vesicles. AgrC(TM5-6C) in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrC(TM5-6C) in detergent micelles. The reconstitution of AgrC(TM5-6C) or AgrC(TM5-6C)-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.