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Scientific article
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English

Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes

Publication date2020-01-21
First online date2020-01-21
Abstract

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.

eng
Keywords
  • ABC transporters
  • DEER
  • EPR
  • Nanobody
  • Spin label
Affiliation Not a UNIGE publication
Funding
Citation (ISO format)
GALAZZO, Laura et al. Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes. In: Proceedings of the National Academy of Sciences of the United States of America, 2020, vol. 117, n° 5, p. 2441–2448. doi: 10.1073/pnas.1913737117
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Article (Published version)
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ISSN of the journal0027-8424
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