IMPORTANCE OF CIS-PROLINE-22 IN THE MEMBRANE-BINDING CONFORMATION OF BOVINE PROTHROMBIN

Upon addition of calcium to the metal-free protein, bovine prothrombin displays a conformational change with behavior of a classic trans- to cis-proline isomerization. The change is accompanied by a decrease of the intrinsic protein fluorescence and is essential to creating the m embrane-binding conformation of prothrombin. This study showed that an identical conformational change was displayed by a peptide correspond ing to residues 1-45 of prothrombin. This peptide contains a single tr yptophan that underwent extensive quenching upon calcium addition, The kinetics were slow (t(1/2) = 2.7 min at 24 degrees C) and displayed a n activation energy of 24 kcal/mol. These properties overlapped precis ely with the behavior of bovine prothrombin fragment I (residues 1-156 ). Consistent with studies on prothrombin and other vitamin K-dependen t proteins that have been modified or truncated, the 1-45 peptide requ ired about 10-fold higher calcium to elicit these behaviors than did f ragment 1. The conformational change was necessary for membrane bindin g by the 1-45 peptide. The only proline in this sequence is at positio n 22. This proline is of the trans configuration in a crystallized for m of calcium-bovine prothrombin fragment 1 [Soriano-Garcia, M., et al. (1992) Biochemistry 31, 2554]. Unless the protein conformational chan ge is based on another behavior, this study showed that biochemical pr operties of the protein are inconsistent with structure solutions, Fur ther studies are needed to reconcile structure/function in membrane as sociation. Proline 22 in bovine prothrombin may constitute a useful bi ochemical marker for the membrane-binding conformation of a vitamin K- dependent protein.