物理化学学报 >> 2019, Vol. 35 >> Issue (5): 546-554.doi: 10.3866/PKU.WHXB201806072

论文 上一篇    

OmpT在Tween-20胶束中折叠的单分子研究

卜佩璇1,2,何晨晖2,3,赵新生1,2,*()   

  1. 1 北京大学化学与分子工程学院化学生物学系,分子动态与稳态结构国家重点实验室,北京分子科学国家研究中心,北京 100871
    2 北京大学生物动态光学成像中心,北京 100871
    3 北京大学生命科学学院,北京 100871
  • 收稿日期:2018-05-09 发布日期:2018-10-19
  • 通讯作者: 赵新生 E-mail:zhaoxs@pku.edu.cn
  • 基金资助:
    国家自然科学基金(21521003);国家自然科学基金(21233002);国家重点基础研究专项经费(2012CB917304)

Single-Molecule Study on the Folding of OmpT in Tween-20 Micelles

Peixuan BU1,2,Chenhui HE2,3,Xinsheng ZHAO1,2,*()   

  1. 1 Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China
    2 Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, P. R. China
    3 School of Life Sciences, Peking University, Beijing 100871, P. R. China
  • Received:2018-05-09 Published:2018-10-19
  • Contact: Xinsheng ZHAO E-mail:zhaoxs@pku.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(21521003);the National Natural Science Foundation of China(21233002);National Key Basic Research Special Foundation of China(2012CB917304)

摘要:

本文用单分子探测研究了外膜蛋白OmpT在Tween-20与十二烷基-β-D-麦芽糖苷(DDM)胶束中的折叠。我们制备了单分散的OmpT,观察到OmpT在不同浓度尿素的Tween-20和DDM胶束中的折叠与去折叠。OmpT在Tween-20胶束中形成的折叠态与其在DDM中折叠形成的天然OmpT结构相似,但稳定性和酶活性均低于天然OmpT。与此相比,在Tween-20中OmpA只在低浓度尿素中折叠,OmpC不折叠。荧光相关光谱(FCS)结果表明折叠的外膜蛋白与去垢剂胶束形成复合体。在β桶折叠机器(BAM)复合体存在时,OmpT比OmpA和OmpC的折叠更加高效。三种外膜蛋白在Tween-20和BAM复合体存在下的折叠结果表明,OmpT比OmpA与OmpC更容易折叠。人们猜测不同性质的外膜蛋白与BAM复合体作用的方式不同,本文的研究结果为这种猜测提供了支持。由于Tween-20常被用于防止单分子实验中的非特异性吸附,本文的结果也提醒人们要注意蛋白与Tween-20相互作用对实验结果的影响。

关键词: 单分子FRET, 荧光相关谱, OmpT折叠, Tween-20, DDM

Abstract:

The cell envelope of gram-negative bacteria consists of the outer membrane (OM), inner membrane (IM), and periplasm. The β-barrel outer membrane proteins (OMPs) embedded in the OM perform diverse and significant functions such as signaling, transporting, and proteolysis. The OMPs of gram-negative bacteria share similar folding pathways with that of mitochondria and chloroplasts. Therefore, the study of the OMP folding mechanism not only provides insights into antimicrobial drug design but also helps elucidate mitochondrial and chloroplast biogenesis. Most knowledge about OMP folding was obtained from ensemble experiments where OMPs were usually at micromolar concentrations and prone to aggregate, which is different from the physiological environment in the cells. Unlike ensemble techniques, single-molecule detection (SMD) can measure OMPs from nano- to picomolar concentrations and prevent aggregation. In this work, we investigated the folding of OmpT, one of the OMPs, in Tween-20 and n-dodecyl β-d-maltopyranoside (DDM) micelles by SMD. We prepared monodisperse OmpT and observed both unfolded and folded OmpT in Tween-20 and DDM micelles under different urea concentrations by single-molecule fluorescence resonance energy transfer (FRET). The folded OmpT in Tween-20 is structurally similar to the native OmpT folded in DDM but exhibits weaker resistance to urea. In contrast, OmpA barely folds and OmpC hardly folds in Tween-20 micelles. We confirmed that folded OmpT forms complexes with detergent micelles and estimated the number of bound Tween-20 and DDM molecules per OmpT by fluorescence correlation spectroscopy. We compared the enzymatic activity of OmpT folded in two detergents with a fluorescent peptide as substrate, and found that the folded form of OmpT in Tween-20 possesses weaker enzymatic activity than that in DDM. We also investigated the folding properties of OmpT, OmpA, and OmpC in the presence of the β-barrel assembly machine (BAM) complex. OmpT folds efficiently in liposome even without the BAM complex; OmpA only folds with the help of the BAM complex; and OmpC does not fold with or without the BAM complex. Based on the comparison of the folding of OmpT, OmpA, and OmpC in detergent micelles and in the presence of the BAM complex, we propose that OmpT has stronger folding tendency than OmpA and OmpC, which supports the idea that the exact role of the BAM complex is dependent on the distinct folding properties of individual OMPs. Since Tween-20 is a widely used reagent to block nonspecific adsorption in SMD experiments, our results also remind people to exercise caution to prevent possible wrong interpretations caused by the interaction between proteins and Tween-20.

Key words: Single-molecule FRET, Fluorescence correlation spectroscopy, OmpT folding, Tween-20, DDM

MSC2000: 

  • O642