Resolving protein conformational ensembles using 19F-NMR and cryo-EM.

Abstract

Detecting and monitoring conformational transitions is essential to understand the protein functional mechanisms. However, it is challenging to observe multiple conformations, especially the lowly populated but often functionally important states. We describe the development of a new 19F NMR protein label with ultra-high sensitivity to conformational changes. The probe achieves chemical shift dispersion of 9 ppm and detects multiple conformational states in a model aspartate transporter GltPh. Technically simple one-dimensional 19F NMR resolved 6 conformational states of the transporter and monitored their population changes triggered by ligands, temperature, and mutations. We further show how 19F NMR can guide sample preparation for cryo-EM imaging to enrich specific structural states in the ensemble, allowing their structural determination. Furthermore, we show a near-quantitative agreement between the spectroscopically observed state populations and the number of particles in the resolved cryo-EM 3D classes. Based on our spectroscopic and structural imaging results, we propose an ensemble description of the transport cycles. The work demonstrates that 19F NMR and cryo-EM could complement each other in studies of dynamic mechanisms of membrane proteins.