CALCIUM ION COORDINATION IN BIOLOGICAL MACROMOLECULES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Calcium is an important secondary messenger involved in several biological processes. These effects are mediated through specific binding of the Ca2+ ions to a variety of regulating proteins. The resting level of Ca2+ in the cytosol of an eukaryotic cell is in the order of 10-100 nM. After stimulation, the concentration of calcium reaches mM levels. This leads to a calcium-dependent conformational change of a variety of Ca2+-binding proteins with the consequence of activating enzymes and other target proteins. The calcium binding protein that we propose to investigate by x-ray absorption spectroscopy is Parvalbumin (PV) that is abundantly found in fast contracting/relaxing muscle fibers. PV is a delayed Ca2+ sink, which plays a major role in calcium homeostasis in the cytosol and is, therefore, usually called a "calcium buffer". PV is also found in different cells types, which undergo rapid and large changes in cytosolic calcium concentration such as GABAergic inhibitory intemeurons. From the EXAFS analysis the existence of strong double-excitation effects has been detected in the first 40 eV of the absorption spectrum. It has been found that a reliable determination of the structural parameters of the fist Ca2+ hydration shell can be obtained only if these effects are properly accounted for in the atomic background. Moreover, in a recent study, both the extended X-ray absorption fine structure spectroscopy (EXAFS) and the large-angle X-ray scattering (LAXS) methods have been used to obtain information about the local structure around the hydrated calcium(II) ion. The choice of PV as study case, is related to the fact that it has only two distinct calcium binding sites and that its structure has been extensively studied by NMR and X-ray crystallography.