Parvalbumins are small Ca2+-binding proteins best known for their cytosolic calcium-buffering activity. The b branch of the parvalbumin (PV) family exhibits a spectrum of divalent ion-binding affinity, with avian thymic hormone (ATH) and rat b-PV at the high- and low-affinity extremes, respectively, and avian parvalbumin 3 (PV3) in the middle. The goal of this project is to elucidate the structural basis for these differing metal ion-binding signatures. Existing data suggest that ion affinity is influenced by determinants outside the metal ion-binding motifs per se; that the interaction between the N-terminal AB region of the molecule and the CD-EF metal ion-binding domain can modulate ion affinity; and that structural differences in the unliganded proteins are responsible in part for the observed variations in affinity. This project has four specific objectives: 1) to identify the amino acid residues in PV3 that attenuate divalent ion affinity relative to ATH; 2) to identify those residues in rat b that attenuate metal ion affinity relative to PV3; 3) to obtain high-resolution structural data on the Ca2+-free forms of rat a (a high-affinity isoform), rat b (a low-affinity isoform), chicken PV3, and, time permitting, chimeric PVs formed from the AB and CD-EF domains of rat a and rat b; and 4) to compare and contrast the backbone dynamics in this same set of proteins. This research is expected to provide substantial insight into protein-ligand interactions, illustrating how the unliganded form of the protein can shape the energetics of ligand binding. The results should have relevance to the nascent field of protein design and engineering.
This project will provide valuable training to graduate and undergraduate students. Individuals involved with this effort will receive exposure to rigorous thermodynamic analysis of protein-ligand interactions and cutting-edge methods for high-resolution protein structure analysis. The data will be integrated into a problem-based graduate physical biochemistry course offered annually by the PI. This ever-evolving course seeks to provide an accessible introduction to physical methods of biomolecular characterization to students with average math and physics skills; to provide these students with an appreciation of the power of these physical tools; to encourage them to consider ways to incorporate these tools into their own research and, ideally, to consider a career in biophysics.
