PROJECT SUMMARY Curved membrane structures such as endocytic pits and trafficking vesicles are essential to cellular physiology. Formation of these structures requires that the proteins involved are able to sense membrane curvature. Two structure-based mechanisms of curvature sensing are known: (i) curvature-matching by crescent-shaped BAR domains and (ii) membrane insertion by amphipathic helices. Recently, the postdoctoral applicant has discovered an additional curvature sensing mechanism that arises not from a specific structural motif, but instead from protein domains that lack a well-defined 3D structure ? intrinsically disordered protein (IDP) domains. How can IDPs sense membrane curvature? Like a random polymer chain, highly water soluble IDPs seek to maximize chain entropy. Tethering polymers to flat surfaces restricts their conformation to a half-plane. In contrast, increasing the curvature of the substrate increases the polymer?s configurational entropy. As such, polymer-like IDPs should display a preference for curved membrane substrates. Because IDP domains are prevalent among endocytic proteins, their ability to sense membrane curvature could strongly impact the initiation and assembly of curved membrane structures. In addition, IDP domains involved in endocytosis are known to form interconnected protein networks, which could further amplify curvature sensing. Preliminary work shows that IDPs have 4-5 times greater affinity for highly curved membrane surfaces in comparison to flatter membranes, which is comparable to structure-based curvature sensing mechanisms. When an IDP and a structured curvature sensing domains were coupled within the same protein, an additional 4-fold increase in curvature sensitivity was observed, suggesting a synergistic relationship among the curvature sensors. The goal of the proposed work is to characterize the ability of IDPs to sense membrane curvature. Work in Aim 1 will evaluate the extent to which IDPs can sense membrane curvature, testing the working hypothesis that IDPs will partition preferentially to highly curved membrane surfaces to maximize chain entropy. Work in Aim 2 will compare curvature sensing by IDPs to sensing by structure-based mechanisms, testing the working hypothesis that entropically-driven curvature sensing by IDPs is comparable in magnitude to the mechanisms used by structured domains. Finally, work in Aim 3 will measure the role of protein networks in amplifying membrane curvature sensitivity, testing the working hypothesis that IDP-containing endocytic proteins cooperatively enhance membrane curvature sensitivity. Current understanding of membrane curvature sensing focusses on specific structural domains. In contrast, this work will be highly significant because it explores the paradigm-shifting idea that proteins lacking a defined structure, IDPs, serve as potent sensors of membrane curvature. The role IDP domains play in curvature sensing and protein network formation is an important, yet unexplored idea in membrane traffic, creating an opportunity to fill a key gap in existing knowledge.