Abstract A fundamental objective in membrane biology is to understand and predict how protein sequences fold and orient in a lipid bilayer. Most studies focus on the membrane protein (MP) and the membrane insertion machinery with little consideration of how lipid environment affects transmembrane domain (TMD) organization. The long-term goal of this proposal is to understand the role of lipid-protein interactions in the assembly, structure and function of MPs. Using a combined molecular genetic and biochemical approach, we established that lipid dependent TMD orientation is dynamic (i.e., can reversibly change) during and after MP assembly in vivo and is independent of other cellular factors in vitro. We proposed the Charge Balance Rule, which includes an acitve role for lipid environment, to explain the dynamic behavior of MPs. We developed a library of lipid mutants of Escherichia coli in which lipid composition can be regulated during or temporally after MP membrane insertion. The physiological significance of membrane lipid bilayer asymmetry is an understudied area. We now add to this library a set of E. coli strains and proteoliposome systems in which membrane lipid bilayer asymmetry can be controlled to determine the role of lipid bilayer asymmetry in MP dynamic organization. Using this set of lipid reagents and native or engineered MPs, we propose three integrated Specific Aims: Aim 1, We will quantify in thermodynamic terms and determine the mechanistic and structural principles underlying the molecular driving forces, as proposed by the Charge Balance Rule, that govern dynamic TMD topology organization as a function of membrane lipid composition and asymmetry. Aim 2, The role of CL in cell functions is not well understood. CL transbilayer asymmetry across any biological membrane is unknown, and there is still no reliable method for estimating its distribution across any membrane. E. coli lacking CL display several phenotypes under aerobic and anaerobic conditions, which would seriously compromise growth in the wild. We will address the role of CL, its transmembrane asymmetry and the three CL synthases in supporting critical cell functions, which will be applicable to the understanding of the importance of CL and cls gene multiplicity in pathogenic Gram-negative bacteria. Aim 3, An essential MP (FtsK) was identified by us associated with a late stage of cell division (a defect in phosphatidylethanolamine-lacking cells) that is topologically responsive to changes in lipid composition and phosphorylation of its extramembrane domains. We will address how changes in lipid composition/asymmetry and phosphorylation/dephosphorylation cycles affect the function of FtsK as an example of a physiological function potentially governed by the Charge Balance Rule. By focusing on interfacial protein-lipid interactions in lipid bilayers with different lipid compositions and asymmetry, measuring electric forces acting on nascent MPs during and after assem...