The omega-loop region of the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes

The hydrophobic omega -loop within the prothrombin gamma -carboxyglutamic a cid-rich (Gla) domain is important in membrane binding. The role of this re gion in membrane binding was investigated using a synthetic peptide, PT-(1- 46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1-46)F4W and PT-( 1-46) bind calcium ions and phospholipid membranes, and inhibit the prothro mbinase complex. PT-(1-46)F4W, but not PT-(1-46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylse rine (PS)-containing vesicles, suggesting Trp ii is located within the moti onally restricted membrane interfacial region, PS-containing vesicles prote ct PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-indu ced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in th e presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous qu enching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles, Fluorescence quenching of membrane-bound PT-(1-46) F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 i s inserted 5 to 7 Angstrom into the bilayer, This report demonstrates that the omega -loop region of prothrombin specifically interacts with PS-contai ning membranes within the interfacial membrane region.