Novel Experimental Strategy for High Resolution AFM Imaging of Membrane-Associated Bacterial Toxins  

作　　者：罗梦麟 邵志峰 沈轶 CZAJKOWSKY Daniel M 孙洁林 

机构地区：[1]School of Biomedical Engineering,Shanghai Jiaotong University [2]Key Laboratory of Systems Biomedicine,Shanghai Jiaotong University

出　　处：《Journal of Shanghai Jiaotong university(Science)》2014年第5期569-573,共5页上海交通大学学报（英文版）

基　　金：the National Natural Science Foundation of China(Nos.991129000,11374207,31370750,21273148 and 11074168)

摘　　要：Bacterial pore-forming toxins(PFTs) are essential virulence factors of many human pathogens. Knowledge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy(AFM) has often been employed to structurally interrogate many membrane proteins, including PFTs, owing to its ability to produce sub-nanometer resolution images of samples under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely-packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O(PFO), as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely-packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sample from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear,bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thereby enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution.Bacterial pore-forming toxins （PFTs） are essential virulence factors of many human pathogens. Knowl- edge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy （AFM） has often been employed to structurally interrogate many membrane proteins, including PFTs, owing to its ability to produce sub-nanometer resolution images of samples under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely-packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O （PFO）, as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely-packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sample from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear, bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thereby enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution.

关 键 词：atomic force microscopy(AFM) perfringolysin O(PFO) bacterial pore forming toxins(PFTs) supported lipid bilayers 

分 类 号：Q71[生物学—分子生物学]