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 acid-rich (Gla) domain is important in membrane binding. The role of this region 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 prothrombinase 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 phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching 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 is inserted 5 to 7 A into the bilayer. This report demonstrates that the omega-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.
Falls
L A
LA
Division of Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
Furie
B C
BC
Jacobs
M
M
Furie
B
B
Rigby
A C
AC
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D010446:Peptide Fragments / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011193:Potassium Iodide / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; C432402:prothrombin (1-46), Trp(4)-; D011193:Potassium Iodide; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin