Tools for probing the physical paralipidome of membrane proteins

Project Summary Membrane (MPs) proteins constitute ~30% of the mammalian proteome and 60% of all drug targets. In mammals, these MPs are solvated by hundreds of distinct lipid types, which can vary dramatically between organelles, across the membrane bilayer, and laterally within a bilayer. Importantly, lipids not only host MPs, but also control their function. Lipids can regulate MP function both as cofactors and ligands, but also through the biophysical properties that arise from lipid collective behaviors. Such properties include fluidity, tension, lipid packing, bilayer thickness, and lateral domains. Further, MPs can selectively recruit local lipid environments that are distinct from the bulk membrane, with varying preferences for lipid types and structural properties (e.g. thickness). These conformation-specific local membrane environments, recently termed paralipidomes, have been reported in isolated and simulated systems, but never directly measured in cells. This proposal intends to overcome this technology gap by covalently modifying MPs with membrane-sensitive fluorophores with the goal of developing and characterizing probes for local MP nano-environments and demonstrating their capabilities in living cell membranes. Cell membrane biophysics relies largely on synthetic fluorescent reporters whose photophysical properties are sensitive to their local membrane environment, e.g. Laurdan, Flipper-TR, and NileRed. While these have been used extensively, in many cases it remains unclear which membrane properties such probes actually sense. In Aim 1, we will employ a combined simulation-experiment methodology to calibrate various sensors to specific membrane structural parameters. We will determine the spatial range sensed by various linker designs and evaluate whether these sensors are affected by coupling to MPs. In Aim 2, we will take a two-pronged approach to demonstrate proof-of-concept for applications of these probes to measuring membrane nano-environments in cells. We will measure how MP paralipidomes evolve through the secretory pathway by coupling sensors to proteins whose transit through the secretory pathway can be tightly controlled. Next, we will measure how the transbilayer asymmetry of lipid distributions in living cell membranes produced local biophysical asymmetries around MPs. We aim to show that MP-coupled reporters can probe the properties of individual leaflets and use these to evaluate biophysical asymmetries at the plasma membrane and in internal organelles. Completion of these aims will provide proof-of-concept for a straightforward, validated, transferrable experimental toolbox for probing local membrane environments around various MPs. Application of these tools will allow access to fundamental open questions about how MPs assemble their local membrane environments and how they can be regulated by their functional paralipidomes.