期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 117, 期 5, 页码 2441-2448出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1913737117
关键词
spin label; nanobody; ABC transporters; EPR; DEER
资金
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy, EXC-2033 [390677874, BO3000/1-2]
- DFG [INST 130/972-1 FUGG]
- Swiss National Science Foundation (SNF) [PP00P3_144823]
- Swiss National Science Foundation (SNF) [PP00P3_144823] Funding Source: Swiss National Science Foundation (SNF)
Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions. We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein.
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