Dynamic Features of Prothrombin Interaction with Phospholipid Vesicles of Different Size and Composition:  Implications for Protein−Membrane Contact

Abstract

The dynamics of prothrombin interaction with membrane vesicles of different size and composition was investigated to ascertain the impact of membrane surface characteristics and particle size on this interaction. Dissociation rates were highly sensitive to membrane composition and varied from about 20/s for membranes of 10% PS to 0.1/s for membranes of 50% PS. Overall affinity also varied by more than two orders of magnitude. Very small differences between prothrombin binding to SUV versus LUV were found. Association with large unilamellar vesicles (LUV of 115 nm diameter) was about 4-fold slower, when expressed on the basis of binding sites, than association with small unilamellar vesicles (SUV, 30 nm diameter) of the same composition. Both reactions proceeded at less than 25% of the collisional limit so that the differences were largely due to intrinsic binding properties. Vesicles of 325 nm diameter showed even slower association velocities. Dissociation rates from LUV were about 2-fold slower than from SUV. Again, these differences arose primarily from intrinsic binding properties. Dissociation conformed to a single first order reaction over a wide range of protein occupancy on the membrane. At very high packing density, the dissociation rate increased by about 2-fold. At equilibrium, prothrombin preferred binding to SUV over LUV by about 2-fold. This very small difference, despite substantial differences in phospholipid headgroup packing and hydrocarbon exposure, appeared inconsistent with an important role for protein insertion into the hydrocarbon region of the membrane. However, prothrombin-membrane interaction may arise from a series of interaction forces that have compensating features at equilibrium. The small differences in prothrombin binding to SUV versus LUV, together with differences in the number of protein binding sites per vesicle, were important to identify mechanisms of substrate delivery to the active site of the prothrombinase enzyme [Lu, Y., & Nelsestuen, G. L. (1996) Biochemistry 35, 8201-8209].

Lupus Antibody Bivalency Is Required to Enhance Prothrombin Binding to Phospholipid

“…The data was well fit to a single rectangular hyperbolae with a dissociation constant, and stoichiometry was 0.68 Ϯ 0.08 M and 0.12 Ϯ 0.01 M, respectively. These parameter values are similar to other estimates of this interaction (19).…”

“…The concentration of prothrombin in the 10-nm diameter shell around the 280-nm diameter phospholipid vesicles was calculated from PL and the stoichiometry of 10,000 molecules/vesicle. Lu and Nelsestuen (19) calculated that 13,000 molecules of prothrombin can bind to 328-nm 75:25 DOPC:DOPS vesicles. K L ( Fig.…”

Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site

“…One possible explanation for enhanced membrane binding is contact of the Tyr 4 side chain with the hydrophobic region of the phospholipid membrane. However, hydrophobic insertion by the -loop has been questioned on many grounds (3,(31)(32)(33)(34)(35). The impact of the Tyr 4 insertion was small (2-fold; ⌬⌬G ϭ 0.4 kcal/mol) compared with the free energy for transfer of a large hydrophobic group such as the phenyl side chain from aqueous solution (to octanol; ⌬⌬G ϭ Ϫ3.6 kcal/mol for ⌬K D ϭ 400-fold) (36).…”

Kinetics of the Interaction of myo1c with Phosphoinositides

“…Hydrodynamic radii of LUVs were measured using dynamic light scattering (DLS; Radii determined by DLS were used to calculate estimated molecular weights (Table 1) of the LUVs as described (22). Stopped-flow Measurements-All measurements were performed in 10 mM HEPES, pH 7, 100 mM NaCl, 1 mM EGTA, and 1 mM DTT at 22 Ϯ 0.1°C.…”