In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels

In situ investigation of membrane proteins is a challenging task. Previously we demonstrated that nitroxide labels combined with pulsed ESR spectroscopy is a promising tool for this purpose. However, the nitroxide labels suffer from poor stability, high background labeling, and low sensitivity. Here we show that Finland (FTAM) and OX063 based labels enable labeling of the cobalamin transporter BtuB and BamA, the central component of the β-barrel assembly machinery (BAM) complex, in E coli. Compared to the methanethiosulfonate spin label (MTSL), trityl labels eliminated the background signals and enabled specific in situ labeling of the proteins with high efficiency. The OX063 labels show a long phase memory time (T_M) of ≈5 μs. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few μm concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.     

电信本地网计费数据联机实时采集系统的设计与分析

阐明了电信本地网计费数据联机实时采集系统的设计原则，介绍了数据链路方式中使用FTAM及CMISE协议实现数据采集的方法，论述了两种联机采集的连接模式，即分散采集与集中采集各自的特点，提供了一种实时监测的手段，及时反映采集错误和机器状态的基本情况，确保系统的安全、稳定运行。