It is the goal of this research to provide a definitive molecular description, by experimental and theoretical techniques, of calcium and magnesium ion binding to prothrombin (human and bovine), its Fragment 1 and appropriate fragment peptides in various states of decarboxylation. Metal binding to prothrombin is thought to be primarily a gamma-carboxyglutamic acid (Gla) event; these aminoacids are prominently displayed along the N- terminus of prothrombin and several other coagulation proteins. We shall (a) conduct a series of condition-consistent metal binding measurements by the technique of equilibrium dialysis, (b) develop a dialysis technique for utilizing the short half-life isotope Mg-28, (c) investigate the Ca2+ ion-selective electrode as a sensitive device for determining low and high concentrations of Ca2+ in the presence and absence of magnesium. In the theoretical studies we will (a) use molecular mechanics and molecular dynamics techniques to find local and global energy minima, respectively, of the 18-23 loop and related Gla containing peptides in the presence and absence of Ca2+ and Mg2+. (b) Use the same techniques as (a) augmented by semiempirical techniques to compute binding of Gla (or malonate) and arginine to (guanidinium) of possible stabilizing salt bridge structures thought to be present in prothrombin. (c) Use distance geometry techniques and the techniques in (a) to interpret experimental 2D- NMR experiments performed on peptides derived or synthesized from the N-terminus regions of Fragment 1 by R.G. Hiskey's group. The theoretical calculations should ultimately provide a good estimate of the metal bound structure of prothrombin. Addendum Further, we will a) investigate the determination of Mg2+ binding with electrodes, b) continue to develop a statistical technique which promises to find the critical number of ligands to effect a biophysical response, c) perform quantum chemical calculations on beta hydroxy aspartic acid models, important in the negative feedback loop of coagulation, d) determine the binding free energy for Ca2+ and Mg2+ to Gla containing models.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL027995-07
Application #
3339416
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
7
Fiscal Year
1991
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
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