Project Summary/Abstract. Intrinsically Disordered Proteins (IDPs) represent approximately 40% of the human proteome and are implicated in a large number of human diseases, including neurological disorders and cancer. Therefore, the biological relevance of IDPs has garnered substantial interest over the last decade. IDPs often interact with curved membranes to form structures that are essential to cellular physiology, such as synaptic and endocytic vesicles. These interactions are facilitated by membrane curvature sensing. The PI recently discovered that IDPs are potent sensors of membrane curvature. This discovery is a substantive departure from the predominant structure-function paradigm, as IDPs lack fixed three-dimensional structure and are often incorrectly assumed to also lack biophysical functionality. The curvature sensitivity of IDPs, as well as many other types of proteins, is normally studied at thermodynamic equilibrium. However, membrane-interacting proteins exist in a dynamic equilibrium between their membrane-bound and membrane-unbound states. It can take minutes to achieve thermodynamic equilibrium between proteins and membranes, but cellular processes, such as cell signaling, occur anywhere between milliseconds to seconds. It is clear from this mismatch in timescales that when thermodynamic equilibrium alone is considered, dynamic information that is pertinent to the timescale of cellular processes is omitted. Little to no literature exists that examines this phenomenon, likely owing to the difficulty associated with achieving such experimental measurements. Thus, dynamic interactions between proteins and curved membrane structures are poorly understood. Using their expertise in quantitative fluorescence microscopy and protein engineering, the goal of the PI's laboratory is to develop and apply techniques and strategies that will allow for direct visualization and characterization of dynamic interactions between IDPs and curved membrane structures. The PI's future research program contains 3 overarching research projects. Work in Project 1 will evaluate the extent to which protein structure influences adsorption and desorption kinetics, testing the working hypothesis that curved membranes affect various protein structures differently. Work in Project 2 will evaluate the impact of protein networks on the binding dynamics of IDPs, answering questions about the influence of protein multivalency on dynamic behavior. Work in Project 3 will develop experimental techniques and strategies that mimic the intracellular environment, answering questions about dynamic interactions between IDPs and curved membrane substrates that occur in the presence of two- dimensional, phase separated protein mixtures on the membrane surface or three-dimensional protein aggregates in the bulk solution. As previously mentioned, our current understanding of the interactions between proteins and curved membranes was derived from systems in which the partitioni...