Remote Determinants of EF-hand Divalent Ion Affinity

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

The parvalbumins - containing two "EF-hand Ca2+-binding motifs - offer an attractive system for examining protein-ligand interactions. Despite extensive homology, parvalbumin (PV) isoforms exhibit disparate metal ion-binding properties. There is compelling experimental evidence that PV divalent ion affinity is influenced by structural features outside the EF-hand motifs. The PV molecule consists of a 70-residue ion-binding domain (the CD-EF domain) and a 40-residue N-terminal AB domain. Others have previously shown that the AB/CD-EF interaction in pike PV is Ca2+-dependent. This project will extend this observation, exploring the hypothesis that the AB domain is a primary modulator of divalent ion-binding behavior. Recombinant AB and CD-EF domains from several a and b parvalbumin isoforms - and select site-specific variants - will be purified and characterized. Following examination of the isolated domains, the energetics of the AB/CD-EF interaction will be delineated in the presence and absence of divalent ions. These issues will be addressed by diverse methods: x-ray crystallography, optical spectroscopy, NMR spectroscopy, analytical ultracentrifugation, surface plasmon resonance, 45Ca2+-binding assays, and titration and scanning calorimetries. Broadly defined, protein-ligand interactions underlie all aspects of protein function - from structure to transport to regulation to catalysis. Importantly, the precise orientation of the coordinating groups in a ligand-binding site can be influenced by structural reorganization events distant from the ligand-binding site. These conformationally mediated "action at a distance" phenomena are among the most intriguing aspects of protein/enzyme action. The parvalbumin molecule - with its juxtaposition of a single EF-hand domain and an autonomous structural element - offers an elegant model system for examining the influence of remote determinants on ligand-binding events and, conversely, the propagation of a ligand-binding signal to neighboring structural elements. Thus, the relevance of these studies extends well beyond structure-affinity correlations in EF-hand proteins.