Construction, properties and specific fluorescent labeling of a bovine prothrombin mutant engineered with a free C-terminal cysteine.
To define the role of phosphatidylserine-induced conformational changes in prothrombin activation during blood coagulation, a recombinant bovine prothrombin was constructed, characterized and shown to have a globally native-like conformation. We introduced a cysteine to replace the penultimate residue (Gly581) of a previously constructed active site mutant, and expressed the double mutant in Chinese hamster ovary cells at the level of 0.6 microgram/ml of cell culture medium. Specific labeling with fluorescein maleimide was accomplished by limited reduction with dithiothreitol to free the engineered cysteine while maintaining the native-like functional properties of the molecule. The average stoichiometry of labeling was 0.84 probe/protein. The location of the probe at the C-terminus was confirmed by proteolysis by native thrombin, by Taipan venom, and by carboxypeptidase Y. Both the double mutant and labeled prothrombin could be activated by snake venoms and the prothrombinase but, as expected, the double mutant meizothrombin did not autolyze as does native meizothrombin. Thus, for the first time, a native-like but specifically labeled prothrombin has been constructed. This molecule will be an essential tool for elucidating the structural role of membranes during prothrombin activation. In addition, the methods described might be usefully applied to labeling of an odd, engineered cysteine in other disulfide bond-containing proteins.
Chen
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Department of Biochemistry and Biophysics CB 7260, University of North Carolina at Chapel Hill 27599, USA.
Lord
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Lentz
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002268:Carboxypeptidases / Q000494:pharmacology; D043402:Cathepsin A; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D011516:Prothrombin / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013917:Thrombin / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism
D018076:DNA, Complementary; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; C023558:prothrombin activator (Taipan venom); D011516:Prothrombin; D013925:Thromboplastin; D002268:Carboxypeptidases; D010447:Peptide Hydrolases; D043402:Cathepsin A; D013917:Thrombin; D003545:Cysteine; D004229:Dithiothreitol; D019793:Fluorescein