Project Summary Membrane proteins regulate the cellular processes by which all organisms survive. Due to this crucial role, membrane proteins are 60% of drug targets. However, improvements to drug design are often impeded by open questions about their mechanisms. A fundamental function of membrane proteins is to transduce information across the membrane by encoding the presence of stimuli in their conformation. Therefore, knowledge of their conformations is required for the missing mechanistic understanding. However, high- resolution structural methods are often limited to individual domains and/or non-native conditions. In contrast, fluorescence-based single-molecule methods are amenable to physiological environments, yet can lack the spatial or temporal resolution required for key conformational changes. Our laboratory recently introduced new methods to improve the temporal resolution of single-molecule spectroscopy and, in the proposed work, will improve the spatial resolution. Investigations into transmembrane behaviors require the full-length protein structure, and thus its native membrane environment. Therefore, we have also developed robust protocols to solubilize membrane proteins from bacteria, plants, and mammals within discoidal lipid bilayers, known as nanodiscs. In initial studies, we used single-molecule spectroscopy and nanodiscs to reveal ligand-induced transmembrane conformational changes for two important receptors, the mammalian epidermal growth factor and the bacterial sugar chemoreceptor Tar. We are now primed to follow the propagation of ligand-induced conformational changes through the receptors and how these changes are controlled by the complex composition and organization of the plasma membrane. Altogether, this NIGMS MIRA application seeks to merge two of my laboratory's primary interests: (1) Developing and applying advanced single-molecule methods for molecular-level insight into protein machinery; and (2) Isolating and interrogating full-length membrane proteins in a near native environment using nanodiscs. Through this combination, we open a window into transmembrane conformational changes and the role of these conformations in cellular processes. Our contributions will impact fields ranging from single-molecule biophysics to cancer biology to microbial signaling.