This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have gathered strong evidence that binding of thrombomodulin (TM) at exosite 1 on thrombin alters the active site of the enzyme towards protein C activation. Our hypothesis is that TM alters thrombin by """"""""dynamic allostery"""""""". This discovery has important implications for the development of better anticoagulants and for understanding the potential of TM fragments in the treatment of disseminated intravascular coagulation. To gain information on the dynamic allostery in the thrombin-TM interaction, we will measure backbone dynamics by NMR. Since the expression of thrombin has only recently been accomplished in E. coli, we are one of the first labs to produce 15N, 13C, 2H-labeled thrombin, we first need to obtain the backbone assignments. The dynamics of free thrombin will be compared active site-inhibited thrombin. Finally, we will deermine how the backbone dynamics of thrombin are affected by TM binding. NMR relaxation experiments will be performed to measure the backbone dynamics of thrombin in the presence and absence of TM fragments. These experiments will reveal backbone dynamics changes that occur within thrombin upon TM binding that are not visible from the crystal structure.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-BCMB-H (40))
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Bhute, Vijesh J; Ma, Yan; Bao, Xiaoping et al. (2016) The Poly (ADP-Ribose) Polymerase Inhibitor Veliparib and Radiation Cause Significant Cell Line Dependent Metabolic Changes in Breast Cancer Cells. Sci Rep 6:36061
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