The functioning of the calcium messenger system is dependent on a series of highly homologous calcium binding proteins that are distinguished by a common helix-loop-helix structural motif in their binding sites. Despite the great structural similarities, these proteins exhibit a wide range of calcium affinity and specificity. One of the principal long term objectives of structural research on this class of protein is to understand the fundamental role of conformation and dynamics in determining specificity and function. The project proposed here will address these issues directly, using the recently developed 1H NMR approach to protein structure determination and the powerful new tool of site-directed mutagenesis. It will provide the first detailed information on conformation and dynamics of a pair of calcium binding sites in both the calcium-free and calcium-loaded forms. The studies will involve calbindin 9K (75 amino acids, M/r=8500). Complete three- dimensional structures will be determined for the apo- and calcium-loaded forms of the protein to address the critical issue of determining the extent of conformational differences that are induced by binding of the calcium ion. Comparative studies of dynamics will be undertaken to determine if the protein mobility and internal accessibility is also affected upon calcium binding. To this end, recombinant DNA technology will be used to produce isotopically labelled proteins in a selective manner, for subsequent analysis of NMR parameters that can be directly correlated with molecular mobility along the backbone and side chains. Site directed mutagenesis will be utilized to produce mutant proteins with altered calcium affinity and specificity. Detailed comparative analysis of the conformation and dynamics of these mutants will provide information on the molecular details that determine calcium affinity, specificity and cooperativity in binding, in terms of the direct interactions of the ligands and long range effects from other parts of the protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM040120-02
Application #
3467082
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
San Diego
State
CA
Country
United States
Zip Code
92037