Neutron scattering determination of the binding of prothrombin to lipid vesicles

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Abstract
Low-angle neutron scattering is used to study the binding of human prothrombin to small single-bilayer vesicles consisting of phosphatidylcholine and phosphatidylserine (1/1 w/w). The radius of gyration of prothrombin indicates that it is an elongated molecule. The vesicles alone were not observed to coalesce, and their molecular weight, outer radius, and average surface area per lipid were respectively (1.6 .+-. 0.32) .times. 106, 114 .+-. 4 .ANG., and 110 .+-. 18 .ANG.2. These values were independent of the presence of calcium and were not altered significantly by prothrombin, which binds reversibly to the vesicle outer surface with its long axis projecting approximately radially forming a 90-.ANG. thick protein shell. From the titration of the protein-vesicle interaction, the apparent dissociation constant of the binding of prothrombin to these vesicles is estimated to be 0.8 .+-. 0.4 .mu.M. At saturation, 57 .+-. 7 prothrombin molecules bind, giving 25 .+-. 6 lipids residues and an area of 2900 .+-. 400 .ANG.2 per prothrombin molecule on the vesicle outer surface. This area is about twice that calculated from a prolate ellipsoid model for prothrombin. However, it is close to the maximum cross-sectional area of fragment 1, the lipid binding region of prothrombin, which is coin-shaped in the high-resolution X-ray structure [Park, C. H., and Tulinsky, A. (1986) Biochemistry 25, 3977-3982]. This similarity suggests that prothrombin binding could be sterically limited.