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Structural comparisons of meizothrombin and its precursor prothrombin in the presence or absence of procoagulant
1Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
Abstract:
A stable form of meizothrombin derived from an active-site (Ser528----Ala) mutant of recombinant bovine prothrombin [Pei et al. (1991) J. Biol. Chem. 266, 9598-9604] has been used to determine the physical properties and conformation of meizothrombin both in solution and when bound to a procoagulant membrane. As determined with quasi-elastic light scattering, meizothrombin and prothrombin had similar molecular dimensions normal to a membrane (9.4 +/- 1.0 nm) and similar binding affinities to procoagulant membranes (1.8 +/- 0.2 microM at 0.4 M NaCl). However, meizothrombin had a greater tendency to form oligomers or aggregates in solution. The enhanced oligomerization of meizothrombin was also evidenced by a high apparent z-weighted molecular weight in equilibrium sedimentation experiments at low spin speeds. However, velocity sedimentation experiments performed at high spin speeds demonstrated the same sedimentation coefficient for meizothrombin (s20,w(0) = 4.7 +/- 0.2 S) as for prothrombin (s20,w(0) = 4.7 +/- 0.15 S). Circular dichroism measurements revealed minor differences in protein secondary structure between meizothrombin and prothrombin either in the presence or in the absence of phospholipid membranes, as reflected in an increased theta 222/theta 208 ratio in meizothrombin relative to prothrombin. The main endotherm of the meizothrombin thermal denaturation profile in a Ca(2+)-containing buffer, as determined by differential scanning calorimetry, was indistinguishable from that of prothrombin. However, in the presence of phosphatidylserine-containing membranes, the peak temperatures of denaturation profiles of meizothrombin were distinct from those of prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Meizothrombin and prothrombin share similar membrane binding and dimensions, but meizothrombin aggregates more in solution. Membrane binding alters meizothrombin’s thermal stability, unlike prothrombin.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Meizothrombin, a precursor to thrombin, plays a role in blood coagulation.
- Understanding meizothrombin's physical properties is crucial for elucidating its function in hemostasis.
- Previous studies have focused on its enzymatic activity, but its structural behavior requires further investigation.
Purpose of the Study:
- To characterize the physical properties and conformation of meizothrombin.
- To compare meizothrombin with prothrombin in solution and when bound to procoagulant membranes.
- To investigate the influence of membrane binding on meizothrombin's structural stability.
Main Methods:
- Utilized quasi-elastic light scattering to determine molecular dimensions and binding affinities.
- Employed equilibrium and velocity sedimentation experiments to assess oligomerization and sedimentation coefficients.
- Conducted circular dichroism spectroscopy to analyze protein secondary structure.
- Applied differential scanning calorimetry to evaluate thermal denaturation profiles.
Main Results:
- Meizothrombin and prothrombin exhibited similar molecular dimensions and binding affinities to procoagulant membranes.
- Meizothrombin showed a greater propensity for oligomerization in solution compared to prothrombin.
- Circular dichroism revealed subtle differences in secondary structure, particularly upon membrane interaction.
- Differential scanning calorimetry indicated distinct thermal denaturation profiles for meizothrombin on phosphatidylserine membranes.
Conclusions:
- Meizothrombin shares structural similarities with prothrombin in terms of membrane interaction but exhibits increased aggregation in solution.
- Membrane binding significantly influences the thermal stability of meizothrombin, suggesting conformational changes.
- These findings provide insights into the structural behavior of meizothrombin and its interaction with phospholipid surfaces during coagulation.