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

doi:10.7503/cjcu20140057

Abstract

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

CLC Number:

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2014, 35(9): 1901.

Figures/Tables 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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