Membrane mimetic thermal proteome profiling (MM-TPP) towards mapping membrane protein-ligand dynamics
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Integral membrane proteins (IMPs) remain the principal target of small-molecule therapeutics, and yet modalities towards probing on and off-target hits against this protein class in a robust, unbiased, and detergent-free manner remain starkly underdeveloped. Previously, we introduced the Peptidisc membrane mimetic (MM) for the water-soluble stabilization of the Escherichia coli membrane proteome and interactome (Carlson et al., 2019). Herein, we implement the Peptidisc into thermal proteome profiling (TPP), enabling for the first time a broad-scale level characterization of membrane protein-ligand interactions while completely circumventing structural perturbations invoked by detergents. Using a library prepared from the whole mouse liver, we determine the influence of ATP and orthovanadate on the thermal stability of IMPs, including pharmaceutically relevant ATP-binding cassette ABC transporters and G-protein coupled receptors. MM-TPP also detects thermal stability changes driven by ATP by-products, where non-canonical ATP binders can be validated with next-generation computational tools. MM-TPP thus offers a robust platform for identifying on- and off-target ligand effects, providing insights into the druggable membrane proteome and its stability as a consequence of changing and often dynamic small molecules.
