ABSTRACT Membrane proteins are responsible for controlling the passage of nutrients, waste, energy and information in and out of cells. They are critical players in physiology and key targets for pharmacological regulation, however, we lack a fundamental understanding of why these proteins are thermodynamically stable in cell membranes. Simply put, why does a greasy protein surface find its greasy protein partner in the greasy lipid bilayer to assemble faithfully into its stable, native structure? In order to investigate this question, the Robertson laboratory has developed a robust and rigorous model system to study equilibrium protein association in membranes based on the reversible dimerization of the CLC-ec1 Cl-/H+ antiporter. Through the development of fluorescence and single-molecule microscopy approaches, it is now possible to experimentally conduct a full thermodynamic analysis of the CLC dimerization reaction in lipid bilayers, yielding the free energy of association (ΔG°) and free energy changes (ΔΔG) due to mutations or different lipid conditions. Recent advances have allowed us to study CLC equilibrium as a function of temperature, enabling a van't Hoff analysis to dissect the thermodynamic changes in enthalpy (ΔH°) and entropy (ΔS°) upon dimerization. In addition, we have developed methods for measuring equilibrium kinetics of CLC dimerization in a tractable manner - in the membrane and in real-time. With this foundation in place, the Robertson lab is primed to build a full molecular model of CLC dimerization in membranes. In the next phase of this project, we will investigate the hypothesis that CLC subunits are driven to associate due to differential solvent dependent driving forces in the associated and dissociated states, and that the transition state involves a critical solvation/de-solvation step. We will investigate this along three distinct aims, by building a theoretical model of the CLC dimerization free energy in membranes using computational approaches (Aim 1), connecting the CLC sequence and structure to dimerization through experimental measurements of thermodynamics and kinetics (Aim 2), and developing a guest-host approach to experimentally and theoretically quantify the impact of mixed lipid composition on protein association equilibria in membranes (Aim 3). Throughout each of these studies, we integrate experiments and theory hand-in-hand, enabling us to make robust connections between physical driving forces and molecular mechanisms. Regardless of the validity of our hypothesis, our studies will provide meaningful quantitative information to the study of membrane proteins in membranes. Ultimately, we expect that the results from these studies will provide a foundation for the field to build new strategies for targeting membrane protein stability and mis-folding based on fundamental physical principles.