UNDERSTANDING THE ROLE OF CONFORMATIONAL DYNAMICS IN PHOSPHOLIPASE Cβ FUNCTION

Phospholipase C β (PLCβ) is an enzyme that plays a key role in cardiovascular function by hydrolyzing phosphatidylinositides (PIs) at the plasma membrane in response to the activation of G protein-coupled receptors at the membrane. PLCβ normally has low basal activity, and its activation is driven by direct binding of the heterotrimeric G protein subunits Gβγ and Gαq. Recent work from our lab and others has shown that the PH domain and first two EF-hands (EF1/2) are conformationally dynamic in solution. This opens up a potential avenue in looking at the dyamics of PLCβ as structures only depict the lipase in a more compact globular shape. We want to address how these known conformational changes PLCβ interacts with the membrane and protein-protein interactions necessary for fuction. In this study, we are using an intramolecular disulfide crosslink to stabilize defined conformational states of PLCβ. These conformational variants will be assessed for their basal activity and their ability to interact with liposomes. In addition, we are laying the initial groundwork to use these variants in single molecule tracking experiments on supported lipid bilayers. These experiments will provide significant insights into how the conformational state of PLCβ contributes to membrane binding and Gβγ stimulation at the membrane interface.