The calcium ion is ubiquitous in biological systems, performing a variety of roles which include functioning as a """"""""second messenger"""""""" in a range of cellular processes. The translation of the Ca 2+ message into metabolic or mechanical responses is mediated by a family of highly homologous proteins that bind Ca 2+ and undergo conformational changes. Besides regulatory functions, members of this family of proteins are involved in Ca 2+ uptake and homeostasis. These proteins have been shown to have important roles in health and disease states, ranging from control of Ca 2+ levels in the body to the distinct differences in the concentration of certain Ca 2+ -binding proteins in tumorigenic and non-tumorigenic cells. The primary long-term objective of this research program is to understand how differences in the sequence, the arrangement of the highly homologous Ca2+-binding domains, and the properties of the protein surface are able to lead to the diversity of responses to Ca2+-binding and consequently, protein function. Although X-ray crystal structures are available for a variety of these proteins with bound ions, there are no direct experimental results establishing the consequences of Ca2+-binding. The NMR approach to protein structure determination in solution is uniquely suited to overcome the limitations posed by the need to crystallize proteins for X-ray analysis; this method is being used to directly determine the structural and dynamical changes associated with Ca 2+ -binding for various members of this family of proteins. The analysis is currently being carried out for one protein from the Ca 2+ uptake/Ca 2+ transport subfamily (calbindin D-9k) and a second protein from the Ca 2+ regulatory subfamily (caltractin). In order to obtain a full understanding of how the diversity of the Ca2+-binding response is achieved, the molecular details of the binding process will also be probed, using a combination of site-directed mutagenesis and NMR.
The specific aims for this granting period are: (1) continue to refine the structural and dynamical description of calbindin D-9k to very high resolution; (2) determine the three-dimensional solution structure of caltractin without bound Ca2+; (3) determine the three-dimensional solution structure of caltractin in the Ca 2+-loaded state and analyze the structural and dynamical consequences of Ca 2+ -binding; (4) probe the molecular details of the Ca2+- binding process in calbindin D-9k using site-directed mutagenesis and NMR.
| Chazin, Walter J (2011) Relating form and function of EF-hand calcium binding proteins. Acc Chem Res 44:171-9 |
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| Hu, Haitao; Sheehan, Jonathan H; Chazin, Walter J (2004) The mode of action of centrin. Binding of Ca2+ and a peptide fragment of Kar1p to the C-terminal domain. J Biol Chem 279:50895-903 |
| Bunick, Christopher G; Nelson, Melanie R; Mangahas, Sheryll et al. (2004) Designing sequence to control protein function in an EF-hand protein. J Am Chem Soc 126:5990-8 |
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| Hu, Haitao; Chazin, Walter J (2003) Unique features in the C-terminal domain provide caltractin with target specificity. J Mol Biol 330:473-84 |
| Veeraraghavan, Sudha; Fagan, Patricia A; Hu, Haitao et al. (2002) Structural independence of the two EF-hand domains of caltractin. J Biol Chem 277:28564-71 |
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