Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Tuesday Speaker Abstracts

Elucidating GPCR Functional Dependence on Plasma Membrane Composition Using Giant Unilamellar Protein-Vesicles Mary Gertrude Gutierrez , Kylee Mansfield, Noah Malmstadt. University of Southern California, Los Angeles, USA. Using an agarose hydration technique for protein incorporation into vesicular bilayers, we elucidate the effects of membrane composition and ordering on G Protein Coupled Receptors (GPRCs). We successfully incorporate GPCRs into model membranes in the form of giant unilamellar protein-vesicles (GUPs). Using this completely in vitro platform we observe that the functional rate of the human serotonin receptor, GPCR 5-HT 1A , and the A2A Adenosine GPCR is dependent on membrane composition and ordering. We use BODIPY-GTPγS as our fluorescent marker to track the irreversible exchange between GDP and GTP on G proteins over time in GUPs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), brain sphingomyelin (BSM), and cholesterol (Chol), as well as synthetic lamellar phase diblock copolymer. Furthermore, using this approach we demonstrate that the incorporated receptors display a biased orientation with the N-terminus located on the exterior (extracellular) and the C- terminus on the interior (cytosolic). Tom1 Modulates Binding of Tollip to Phosphatidylinositol 3-Phosphate via a Coupled Folding and Binding Mechanism Shuyan Xiao 1 , Mary K. Brannon 1 , Xiaolin Zhao 1 , Kristen I. Fread 1 , Jeffrey F. Ellena 2 , John H. Bushweller 2 , Carla V. Finkielstein 3 , Geoffrey S. Armstrong 4 , Daniel G. Capelluto 1 . 1 Virginia Tech, Blacksburg, VA, USA, 2 University of Virginia, Charlottesville, VA, USA, 4 University of Colorado at Boulder, Boulder, CO, USA. 3 Virginia Tech, Blacksburg, VA, USA, Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism. NMR data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N-terminus when bound to Tom1 GAT through high affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Tom1 GAT is also able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors Tollip’s release from endosomal membranes, allowing Tollip to commit to cargo trafficking.

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