The overall objectives of this project are to begin studies to understand the vitamin K cycle, which is important for blood coagulation, bone formation, and probably other physiological processes. The project is divided into four specific aims; for the first three we will continue our work on the structure and function of the vitamin K-dependent enzyme gammaglutamyl carboxylase. We will use alanine-scanning mutagenesis for the first two aims: to determine the propeptide and vitamin K binding sites on the carboxylase molecule and to characterize a naturally-occurring carboxylase gene mutation found in a patient. Since the structure of the carboxylase molecule is essential to understanding its function our third aim will be to analyze both topology and 3-dimensional structure. The method of topological analysis we have chosen is particularly well-suited to integral membrane proteins such as the carboxylase, as is our method of electron diffraction to determine 3-dimensional structure. Finally, for our fourth specific aim, we will focus on another enzyme important in the vitamin K cycle, the vitamin K epoxide reductase. We will begin work to purify and characterize this protein, about which little is currently known. One of our long-term goals is to combine the carboxylase and vitamin K epoxide reductase, along with any other essential components, in an in vitro system to start to reconstitute the entire vitamin K cycle. These studies have significant clinical relevance in that they could lead to improved therapies for blood coagulation and thrombosis disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL048318-08
Application #
6043783
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1992-04-01
Project End
2001-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
8
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Parker, Christine H; Morgan, Christopher R; Rand, Kasper D et al. (2014) A conformational investigation of propeptide binding to the integral membrane protein ?-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry. Biochemistry 53:1511-20
Tie, J-K; Jin, D-Y; Tie, K et al. (2013) Evaluation of warfarin resistance using transcription activator-like effector nucleases-mediated vitamin K epoxide reductase knockout HEK293 cells. J Thromb Haemost 11:1556-64
Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W (2012) Human vitamin K epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms. J Biol Chem 287:33945-55
Tie, Jian-Ke; Jin, Da-Yun; Straight, David L et al. (2011) Functional study of the vitamin K cycle in mammalian cells. Blood 117:2967-74
Morgan, Christopher R; Hebling, Christine M; Rand, Kasper D et al. (2011) Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs. Mol Cell Proteomics 10:M111.010876
Higgins-Gruber, Shannon L; Mutucumarana, Vasantha P; Lin, Pen-Jen et al. (2010) Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone, and glutamate substrate binding on the structure and function of {gamma}-glutamyl carboxylase. J Biol Chem 285:31502-8
Hebling, Christine M; Morgan, Christopher R; Stafford, Darrel W et al. (2010) Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry. Anal Chem 82:5415-9
Tie, Jian-Ke; Zheng, Mei-Yan; Hsiao, Kuang-Ling N et al. (2008) Transmembrane domain interactions and residue proline 378 are essential for proper structure, especially disulfide bond formation, in the human vitamin K-dependent gamma-glutamyl carboxylase. Biochemistry 47:6301-10
Gui, Tong; Reheman, Adili; Funkhouser, William K et al. (2007) In vivo response to vascular injury in the absence of factor IX: examination in factor IX knockout mice. Thromb Res 121:225-34
Davis, Charles H; Deerfield 2nd, David; Wymore, Troy et al. (2007) A quantum chemical study of the mechanism of action of Vitamin K carboxylase (VKC) III. Intermediates and transition states. J Mol Graph Model 26:409-14

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