Subjects: Biology >> Biophysics submitted time 2016-05-12
Liu, Bei
Xue, Yanhong
Zhao, Wei
Chen, Yan
Fan, Chunyan
Gu, Lusheng
Zhang, Yongdeng
Zhang, Xiang
Sun, Lei
Huang, Xiaojun
Ding, Wei
Sun, Fei
Ji, Wei
Xu, Tao
Liu, Bei
Gu, Lusheng
Zhang, Yongdeng
Xu, Tao
Zhao, Wei
Chen, Yan
Abstract： We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.
Peer Review Status:
Awaiting Review
Subjects: Biology >> Biophysics submitted time 2016-05-12
Wang, Xu-Ying
Wang, Dian-Bing
Bi, Li-Jun
Zhang, Xian-En
Wang, Xu-Ying
Zhang, Ji-Bin
Zhang, Zhi-Ping
Ding, Wei
Abstract： S-layer proteins create a cell-surface layer architecture in both bacteria and archaea. Because S-layer proteins self-assemble into a native-like S-layer crystalline structure in vitro, they are attractive building blocks in nanotechnology. Here, the potential use of the S-layer protein EA1 from Bacillus anthracis in constructing a functional nanostructure is investigated, and apply this nanostructure in a proof-of-principle study for serological diagnosis of anthrax. EA1 is genetically fused with methyl parathion hydrolase (MPH), to degrade methyl parathion and provide a label for signal amplifi cation. EA1 not only serves as a nanocarrier, but also as a specific antigen to capture anthrax-specific antibodies. As results, purified EA1-MPH forms a single layer of crystalline nanostructure through self-assembly. Our chimeric nanocatalyst greatly improves enzymatic stability of MPH. When applied to the detection of anthrax-specific antibodies in serum samples, the detection of our EA1-MPH nanostructure is nearly 300 times more sensitive than that of the unassembled complex. Together, it is shown that it is possible to build a functional and highly sensitive nanosensor based on S-layer protein. In conclusion, our present study should serve as a model for the development of other multifunctional nanomaterials using S-layer proteins.
Peer Review Status:Awaiting Review
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