Adsorption of bovine prothrombin to spread phospholipid monolayers

Abstract

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.

The immediate‐early oncoproteins Fra‐1, c‐Fos, and c‐Jun have distinguishable surface behavior and interactions with phospholipids

“…Second, the specific affinity of c‐Fos towards LE phase and anionic phospholipids was also found for prothrombin54 and explained by hydrophobic interactions 55. The degree of protonation is probably involved in the response of Fra‐1 and c‐Fos to charge and phase, but electrostatics may play a minor role in their interaction with PS and PIP 2 56; the protein molecular rearrangement dependent on packing may participate more strongly in the affinity for specific lipid phases.…”

Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa

“…This is surprising, because binding of Gla-containing proteins to PS-containing membranes has long been seen as mediated by a Ca 2ϩ -induced conformational change of the Gla domain (43)(44)(45)(46). It may be that binding of Gla-containing proteins to a PS-containing membrane involves adsorption of the Ca 2ϩ -conformation of the Gla domain to a membrane surface (31,(47)(48) rather than recognition of individual PS molecules by specific binding sites. By contrast, the C6PS-induced conformational change that we see in the absence of Ca 2ϩ does involve a single C6PS molecule (Table III).…”

Probing the Structural Changes in the Light Chain of Human Coagulation Factor VIIa Due to Tissue Factor Association

“…The Gla domain is thought to be responsible for lipid binding (Pollock et al, 1988;Wildgoose et al, 1992). The interaction of the Gla domain with the lipid surface has been proposed to be via calcium bridging (Nelsestuen, 1988) or through insertion of hydrophobic residues to the lipid surface (Ellison and Castellino, 1997). Although there is a slight Gla-EGF1 reorientation in solution with TF absent, the spatial arrangement that may be provided for lipid binding appears similar in TF-free solution and TF-bound crystal structures.…”