Cholesterol plays a multifaceted role in the regulation of animal cell membranes and an inability to control excessive cholesterol levels has been implicated in many human diseases including atherosclerosis, fatty liver disease and cardiovascular disease. Due to its dual nature (essential but toxic in excess), cellular cholesterol content is tightly regulated. Cellular cholesterol levels are controlled through the feedback regulation of the Scap- SREBP pathway, which is comprised of a collection of membrane proteins in the endoplasmic reticulum (ER). The primary proteins involved in the cholesterol sensing pathway include several integral membrane proteins: Scap, transcription factors named Sterol Regulatory-Element Binding Proteins (SREBPs), and Insigs. When ER cholesterol levels drop below a threshold, Scap transports SREBPs to the Golgi, where proteolytic activation of SREBPs results in cholesterol synthesis and uptake. When ER cholesterol levels rise above a threshold, Insig binds Scap and prevents Scap-SREBP from traveling to the Golgi, terminating further cholesterol synthesis and uptake. The central question of this research proposal is: how does Scap sense changes in ER membrane cholesterol? Understanding the structural and biophysical role of cholesterol’s influence on Scap will uncover the molecular mechanism of feedback regulation of the Scap-SREBP pathway. Preliminary work has yielded a Scap-Insig complex cryo-EM reconstruction enabling the modeling of cholesterol at the Scap-Insig interface. While investigating Scap residues involved in the cholesterol interaction, multiple residues were also identified to play a role in SREBP-transport. Therefore aim 1 proposes to determine the cryo-EM structures of Scap in a SREBP-transporting conformation and in the presence of cholesterol-saturated detergent micelles or reconstituted into cholesterol-rich lipid bilayer nanodiscs. A complete set of structures will enable deciphering of the mechanism of how cholesterol influences Scap to stop transporting SREBPs and instead allow for Insig- binding. Recent moderate-resolution (~4 angstrom) structures of Scap and the Scap-Insig complex suggest a large conformational change in the luminal extramembranous globular domain (L1L7) of Scap between the Insig- free and Insig-bound states. Therefore aim 2 seeks to study how cholesterol influences the movement of the L1L7 domain by developing and implementing a structure guided single-molecule FRET (Fluorescence Resonance Energy Transfer, smFRET) assay to assess the conformations of purified Scap in the presence of cholesterol. Preliminary studies have generated and confirmed the cellular regulation of Scap constructs meant for labeling experiments. To understand the progression of the conformational changes seen in the current structures, movement of the L1L7 domain will be measured at high-speed resolution using smFRET. Together these studies will provide a better structural and biophysical understanding of...