Abstract
Thrombin is a serine protease central to regulation of the blood clotting cascade. Active wild-type thrombin acts as a procoagulant and cleaves the zymogen fibrinogen to produce fibrin, which forms the scaffold structure of a blood clot. Thrombin contains many intrinsically disordered loops near it's active site and exosites that are believed to facilitate specificity of thrombin towards it's substrates through small time scale entropic movements when cofactors are bound. To study the dynamic properties of these mutants, we produced W215I and W215A mutants of human thrombin and carried out hydrogen-deuterium exchange (HDXMS) experiments using the Synapt G2Si instrument with ion mobility separation. This new instrument gave much higher coverage of the thrombin sequence and we were able to discover where the dynamics of the thrombin had changed with the mutations. Amide exchange in the 220s loop was strongly increased in the W215A mutant as compared to wild-type, and amide exchange in the beta-sheet leading up to position 215 (res. 209-214) was markedly decreased. These results help to explain why mutants at Trp215 have markedly different substrate specificity.
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