| Seven plasma proteins, mainly involved in blood clotting, depend on vitamin K for their biosynthesis. The vitamin K-dependent structure, ;Investigations with amino-terminal peptides suggested that the membrane contact region resided between residues 4-38. Comparisons of amino acid sequences and membrane affinities with human and bovine proteins allowed proposal of a new membrane contact model. The range of membrane affinities was mostly due to amino acid variations at residues 11, 33, and 34.;To test the new model for membrane contact, mutants of bovine and human protein C were generated. The presence of proline at position 10 (homologous to position 11 in bovine prothrombin) lowered the membrane affinity and enzyme activity. Replacing the proline with histidine produced enhanced membrane-binding and enzyme activity. This supported the suggestion of a central role for residue 11 in creating variable membrane-binding properties.;Observations of the new membrane contact model suggested that these proteins have an electropositive care (protein-bound calcium ions) surrounded by an envelope of electronegative amino acids. The range of membrane affinities appeared to correspond to the charge on residues 11, 29, 33, and 34. Modification of position 33 and 34 by site-directed mutation adjusted the electrostatic properties to produce an enhanced bovine protein C molecule.;The strategy of improving biological function by manipulating the membrane-binding site was also tested on human factor VII. A variant factor VII (VII-Q10E32) was produced. Biochemical studies revealed a significant enhancement relative to wild-type factor VII. It was speculated that the VII-Q10E32 may provide more effective treatment of bleeding episodes than wild-type factor VIIa.;Overall, this work has shown that manipulation of the membrane contact site to alter function is applicable to all of the vitamin K-dependent plasma proteins and, consequently, can provide important research and medical applications. |
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