Project summary We propose purchasing a High-Performance Liquid Chromatography (HPLC) system to place in-line with an existing multi-angle laser light scattering instrument in the Harms lab. This will allow us to precisely characterize the oligomeric states of TLR4 (Toll-like receptor 4) and its associated proteins. The overall goal of the funded parent project is to dissect the evolution of the TLR4 protein complexes over the last 400 million years. TLR4 is an important driver for inflammation in both healthy contexts (e.g, response to infection, wound healing) and pathological cancers (e.g., chronic inflammation, cancer). It interacts with multiple protein partners as part of these functions, including MD-2, CD14, and S100A9. To understand the evolution and mechanism by which the TLR4 complex has its effects, we need sharp tools to dissect the interactions between these proteins. All three aims of the parent project will benefit from the requested instrumentation. The first two aims require understanding how TLR4 binds to different agonist molecules and oligomerizes in response; the third aim looks the evolution of the key support molecule CD14, which directly binds TLR4. The ability to directly measure protein-protein interactions and oligomerization is critical to achieving these aims.