Alpha and Beta Parvalbumins: Functional Consequences of Divergent Tertiary Interactions

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9603877 Henzl Despite their general similarity, parvalbumin (PV) isoforms exhibit substantial physical and functional diversity. This project will examine three potential mechanisms for achieving isoform-dependent variation. The importance of tertiary interactions between the C-terminus and residues 26- 36 will be examined in select site-specific PV variants -- employing differential scanning calorimetry, Ca2+-binding measurements, titration calorimetry, and 15N NMR relaxation measurements. To assess the impact of overall electrostatic free energy on metal ionbinding behavior, the consequences of the S55D and G98D mutations on Ca2+- and Mg2+- binding affinity will be examined in PV isoforms differing widely in their isoelectric points. Finally, the minimal sequence requirements for the metal ion-facilitated association of CPV3, an avian -PV, will be examined. The energetics of complexation will be investigated by analytical ultracentrifugation and scanning calorimetry. Low- resolution structural information on the trimer will be obtained by antibody reactivity and chemical crosslinking studies. Detailed structural data will be obtained via x- ray crystallography and/or NMR. Contemporary protein research seeks to identify the physical principles that dictate protein structure and function. The parvalbumins offer an attractive system for examining these issues. The vertebrate-specific family of small Ca2+ binding proteins illustrates the application of the same basic protein architecture to the solution of distinct biological problems -PVs play a rather passive role in maintaining cytosolic Ca2+ levels below the toxic threshold. By contrast, in higher vertebrates, -PVs are utilized for highly specialized signaling roles. This functional variation is accompanied by corresponding variation in physical properties. For example, -PVs display greater structural rigidity than -PVs. Thus, one objective of this project is to investigate the molecular basis for this difference. Additionally, whereas all -PVs bind Ca2+ very tightly, the -PVs display highly variable affinity for the ion. Therefore, a second objective of this project is to assess the impact of the overall charge on metal ion-binding properties. Lastly, although parvalbumins are widely viewed as non-associating monomeric proteins, the avian -PV CPV3 forms a trimeric complex in the presence of Ca2+ or Mg2+. Thus, a third goal of this project is to identify the structural idiosyncrasies responsible for this self- complementarity. This work will illuminate demonstrably successful strategies for tailoring the physical stability and ligand-binding properties of macromolecules through subtle sequence alterations. The knowledge gained from these studies may assist in achieving the long-term goal of manipulating existing biological activities and designing new ones.