Project Summary Integral membrane proteins reside in the biological membrane where they function and intimately interact with lipid molecules. The membrane environment is dynamic and composed of a rich chemical diversity of lipids. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in modulating the structure and function of membrane proteins. Although a small subset of examples exist that provide insight into membrane protein-lipid interactions, how individual lipid molecules influence the structure and function of membrane proteins on the molecular level remains poorly understood. What determines the selectivity of membrane proteins towards lipids? How important is the lipid chemistry such as lipid tail length, stereochemistry, and position of unsaturated double bonds in protein-lipid interactions? Addressing these fundamental questions is hindered not only by identifying the lipids that bind avidly to membrane proteins but also the biophysical characterization of protein-lipid interactions. Herein, this proposal seeks to develop and apply a highly innovative and integrative approaches to better understand how lipids impact the structure and function of membrane proteins. Our first objective is the development of integrative methods, combining lipidomics with native mass spectrometry (MS), to identify specific protein-lipid interactions from natural extracts by using (i) progressive washes of the immobilized membrane proteins and (ii) lipid exchange within empty and membrane protein loaded nanodiscs. In our second objective, native MS technology will be used to biophysically characterize individual lipid binding events to membrane proteins, providing insight into affinity and selectivity. Moreover, MS approaches of membrane proteins in nanodiscs will be employed to glean insight into lipids enriched around membrane proteins. These new methods will identify lipids that avidly associate with the target membrane protein, providing a roadmap for our third objective focused on understanding how tightly bound lipids affect function and structure of membrane proteins. Membrane proteins devoid of any contaminating lipids will be reconstituted into liposomes and nanodiscs in the presence and absence of a tightly bound lipid. Structural and functional studies will lead to visualization of lipid binding sites and structural and functional changes induced by the bound lipids. Observations from structural and functional studies will then be rigorously examined with mutational studies. Taken together, the results and outcomes from our proposed studies are anticipated to have a significant impact in our understanding of membrane protein-lipid interactions and, more generally, how changes in the biological membrane may regulate membrane protein physiological function.