It is the goal of this research plan to provide a definitive study of the molecular role of metal ion binding (Ca++, Mg++, Eu+++, Eu++) to the blood clotting proteins prothrombin fragment 1 and prothrombin 1-39 from human and bovine sources. Both experimental and theoretical techniques will be used. Experimentally our plan is to: (1) conduct as series of conditions - consistent binding measurements by the techniques of equilibrium dialysis, ultrafiltration and atomic absorption; (2) prepare, characterize and utilize Eu++ as a potential luminescent Ca++ probe; (3) utilize monoclonal antibody binding to specific metal-ion induced conformations of prothrombin fragment 1 so as to determine affinity constants and thus make possible quantitative comparison (between metals) of metal ion induced states. Theoretically our plan is to (1) utilize the binding curves determined experimentally to distinguish between possible cooperativity models; (2) determine by ab initio quantum mechanical methods how Ca++ (and Mg++) binds at the molecular level to Gla and Gla-Gla and their analogs; (3) carry out energy minimization calculations for prothrombin 1-39 so as to find the low energy forms in the presence of metal binding. The relationship between the theoretical concept of cooperativity and experimental binding data is at the core of understanding the energetics and dynamics of protein function. Our overall goal is to clarify this relationship by a study on an important health-related system that we feel can be quantitatively defined.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL027995-03
Application #
3339413
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1983-05-01
Project End
1987-11-30
Budget Start
1985-05-01
Budget End
1987-11-30
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Arts and Sciences
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Chen, Z; Blandl, T; Prorok, M et al. (1998) Conformational changes in conantokin-G induced upon binding of calcium and magnesium as revealed by NMR structural analysis. J Biol Chem 273:16248-58
Li, L; Darden, T A; Freedman, S J et al. (1997) Refinement of the NMR solution structure of the gamma-carboxyglutamic acid domain of coagulation factor IX using molecular dynamics simulation with initial Ca2+ positions determined by a genetic algorithm. Biochemistry 36:2132-8
Li, L; Darden, T; Foley, C et al. (1995) Homology modeling and molecular dynamics simulation of human prothrombin fragment 1. Protein Sci 4:2341-8
Deerfield 2nd, D W; Pedersen, L G (1995) The first solvation shell of magnesium and calcium ions in a model nucleic acid environment: an ab initio study. J Biomol Struct Dyn 13:167-80
Deerfield 2nd, D W; Fox, D J; Head-Gordon, M et al. (1995) The first solvation shell of magnesium ion in a model protein environment with formate, water, and X-NH3, H2S, imidazole, formaldehyde, and chloride as ligands: an Ab initio study. Proteins 21:244-55
Cabaniss, S; Deerfield 2nd, D W; Monroe, D M et al. (1995) Is Ca(II) ion binding to prothrombin fragment 1 intrinsically cooperative, or is the cooperative binding accounted for by self-association? Blood Coagul Fibrinolysis 6:464-73
Frech, M; Darden, T A; Pedersen, L G et al. (1994) Role of glutamine-61 in the hydrolysis of GTP by p21H-ras: an experimental and theoretical study. Biochemistry 33:3237-44
York, D M; Darden, T A; Pedersen, L G et al. (1993) Molecular dynamics simulation of HIV-1 protease in a crystalline environment and in solution. Biochemistry 32:1443-53
York, D M; Darden, T A; Pedersen, L G et al. (1993) Molecular modeling studies suggest that zinc ions inhibit HIV-1 protease by binding at catalytic aspartates. Environ Health Perspect 101:246-50
Foley, C K; Pedersen, L G; Charifson, P S et al. (1992) Simulation of the solution structure of the H-ras p21-GTP complex. Biochemistry 31:4951-9

Showing the most recent 10 out of 37 publications