Remote Determinants of EF-hand Divalent Ion Affinity

含有两个EF-手Ca 2+结合基序的小清蛋白为检查蛋白质-配体相互作用提供了一个有吸引力的系统。尽管具有广泛的同源性，但小清蛋白（PV）同种型表现出不同的金属离子结合特性。有令人信服的实验证据表明，PV二价离子亲和力的影响EF-手图案以外的结构特征。PV分子由70个残基的离子结合结构域（CD-EF结构域）和40个残基的N-末端AB结构域组成。其他人先前已经表明，在派克PV中的AB/CD-EF相互作用是Ca 2+依赖性的。本项目将扩展这一观察，探索AB结构域是二价离子结合行为的主要调节剂的假设。将纯化和表征来自几种a和B小清蛋白同种型的重组AB和CD-EF结构域-以及选择位点特异性变体。在检查分离的结构域之后，将在存在和不存在二价离子的情况下描绘AB/CD-EF相互作用的能量学。这些问题将通过不同的方法来解决：X射线晶体学，光谱学，NMR光谱学，分析超离心，表面等离子体共振，45 Ca 2+结合测定，滴定和扫描量热法。 从广义上讲，蛋白质-配体相互作用是蛋白质功能的所有方面的基础-从结构到运输到调节再到催化。重要的是，配体结合位点中的配位基团的精确取向可以受到远离配体结合位点的结构重组事件的影响。这些构象介导的“远距离作用”现象是蛋白质/酶作用最有趣的方面之一。小清蛋白分子-与其并列的一个单一的EF-手域和一个自主的结构元件-提供了一个优雅的模型系统，用于检查远程决定因素对配体结合事件的影响，相反，配体结合信号的传播到邻近的结构元件。因此，这些研究的相关性远远超出了EF-手蛋白的结构-亲和力相关性。

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.