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 membrane-binding conformation of prothrombin. This study showed that an identical conformational change was displayed by a peptide corresponding to residues 1-45 of prothrombin. This peptide contains a single tryptophan that underwent extensive quenching upon calcium addition. The kinetics were slow (t1/2 = 2.7 min at 24 degrees C) and displayed an activation energy of 24 kcal/mol. These properties overlapped precisely with the behavior of bovine prothrombin fragment 1 (residues 1-156). Consistent with studies on prothrombin and other vitamin K-dependent proteins that have been modified or truncated, the 1-45 peptide required about 10-fold higher calcium to elicit these behaviors than did fragment 1. The conformational change was necessary for membrane binding by the 1-45 peptide. The only proline in this sequence is at position 22. This proline is of the trans configuration in a crystallized form of calcium-bovine prothrombin fragment 1 [Soriano-Garcia, M., et al. (1992) Biochemistry 31, 2554]. Unless the protein conformational change is based on another behavior, this study showed that biochemical properties of the protein are inconsistent with structure solutions. Further studies are needed to reconcile structure/function in membrane association. Proline 22 in bovine prothrombin may constitute a useful biochemical marker for the membrane-binding conformation of a vitamin K-dependent protein.
Evans
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TC
Department of Biochemistry, University of Minnesota, St. Paul, 55108, USA.
Nelsestuen
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GL
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism
D008081:Liposomes; D010446:Peptide Fragments; D011516:Prothrombin; D011392:Proline; D002118:Calcium