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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. [ABSTRACT FROM AUTHOR]