Soluble proteins are key mediators of many biochemical signaling pathways via direct interaction with the lipid bilayer and via membrane-bound receptors. Components of the cell membrane are involved in many important biological processes including blood coagulation, viral infection, and signal transduction, and as such, they are common targets of therapeutic agents. Therefore, the development of analytical approaches to study interactions at the cell membrane is of critical importance. This proposal integrates two key technologies, silicon photonic microring resonator arrays and phospholipid bilayer Nanodiscs, which together allow multiplexed screening of soluble protein interactions with lipid and membrane-embedded targets. Microring resonator arrays are an intrinsically multiplexable, label-free analysis platform that has previously been applied to studying protein-protein, protein-nucleic acid, and nucleic acid-nucleic acid interactions. Nanodiscs are protein-stabilized lipid assemblies that represent a convenient construct to mimic the native phospholipid bilayer, investigate the effects of membrane composition, and solubilize membrane-embedded targets. Fused together, these technologies will offer an unsurpassed capacity to interrogate the biophysical modalities of membrane-ligand interactions in high-throughput, multiplexed, and information rich assay formats. The biological motivation for the proposed work is blood coagulation, which is a critical regulatory process that is a target for therapeutic intervention i thrombosis and bleeding disorders. In addition to protein- membrane protein interactions, the assembly of coagulation factors at the cell membrane surface is additionally regulated by protein-lipid interactions, and in particular interactions with anionic lipids that are critically-dependent upon divalent metal ions. However many details of protein-lipid interactions in blood clotting remain poorly understood, and an improved fundamental understanding might reveal new therapeutic strategies for life-threatening thrombotic diseases. Herein we propose to shed light onto complex lipid- and cation-dependent binding of blood coagulation factors (Factors VIIa and X; fVIIa and fX, respectively) and clotting inhibitors (activated protein C; aPC) through the use of multiplexed arrays of microring resonators functionalized with Nanodiscs that present variable, yet well-defined lipid composition. We also will implement a novel solution-phase gradient maker to dynamically control the concentration and identity of metal cations in solution while simultaneously monitoring the effects of these changes on protein-lipid interactions in real time. Together, these studies will provide unprecedented access to high throughput and multivariate interrogation of blood coagulation processes at model interfaces, and more generally will validate the integration of Nanodiscs and microring resonator arrays for highly multiplexed studies of interactions at the cell membrane.
Soluble protein interactions at the cell membrane mediate a number of important biological processes, including blood coagulation, viral infection, and signal transduction, and as such, they are common targets of therapeutic agents. We propose to fuse together two enabling technologies, lipid bilayer Nanodiscs and silicon photonic sensor arrays, to allow an unprecedented capacity to interrogate the biophysical modalities of membrane-ligand interactions in high-throughput, multiplexed, and information rich assay formats. We apply this platform to the elucidation of synergies within the blood coagulation cascade, but a more general objective is to validate a powerful approach for highly multiplexed studies of biomolecular interactions occurring at the cell membrane interface.
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