Many intracellular processes are regulated by a transient change in the concentration of Ca2+ ions. The effect of Ca2+ is mediated by proteins that bind Ca2+ specifically and assume a new conformation, which then enables them to bind to target proteins, including some enzymes, thereby affecting their function.
The aim of this project is to understand molecular mechanisms by which the two best characterized Ca2+-binding regulatory proteins, calmodulin (CaM) and troponin C (TnC), recognize and modulate their targets in a Ca2+-dependent way. Local and global conformational changes induced in CaM and TnC by Ca2+ and by interactions with selected targets - whole proteins (phosphodiesterase, calcineurin, neuromodulin, troponin I) and peptides that mimic the interaction sites - will be characterized. Mutants of human liver CaM and rabbit skeletal TnC will by synthesized with Cys residues at key positions. Changes in distances between the Cys side chains will be monitored following their labeling with chromophores suitable for fluorescence energy transfer measurements. The functional significance of specified conformational transitions in CaM and TnC will be assessed by evaluating the target-modulating ability of mutants in which the movement of selected protein segments is restricted by a disulfide bond. Sites of interaction with target molecules on CaM and TnC will be characterized in detail with a view to defining determinants of specificity. Mutants will also be used to assess the contribution of some hydrophobic and ionic groups at specific positions to the strength of interaction and to the modulatory activity. These studies will contribute to a better understanding of the mechanism of Ca2+ signal transduction by CaM, TnC and other Ca2+-binding proteins. The detailed description o the interaction interfaces and definition of determinants of specificity will be important for identification of physiological targets for other members of the rapidly growing family of Ca2+-binding proteins.
