Remote Determinants of EF-hand Divalent Ion Affinity

含有两个“EF-手钙结合基序”的小蛋白为研究蛋白质-配体的相互作用提供了一个有吸引力的系统。尽管有广泛的同源性，但小白蛋白(PV)亚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二价离子亲和力受到EF-Hand基序外部结构特征的影响。PV分子由一个70个残基的离子结合结构域(CD-EF结构域)和一个40个残基的N-末端AB结构域组成。其他研究表明，PICE PV中的AB/CD-EF相互作用是依赖于钙离子的。这个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器的假设。来自几种a和b小白蛋白异构体的重组AB和CD-EF结构域-以及部分特定部位的变体-将被提纯和鉴定。在研究了孤立的磁区后，AB/CD-EF相互作用的能级将在存在和不存在二价离子的情况下被描绘出来。这些问题将通过不同的方法解决：X射线结晶学、光学光谱、核磁共振光谱、分析超速离心法、表面等离子体共振、45Ca2+结合分析以及滴定和扫描量热法。从广义上讲，蛋白质-配体相互作用是蛋白质功能的各个方面的基础--从结构到运输，再到调节再到催化。重要的是，配位基团在配基结合部位的精确定位会受到远离配基结合部位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/酶作用最耐人寻味的方面。小白蛋白分子--它的单个EF-Hand结构域和一个自主结构元素并列在一起--提供了一个优雅的模型系统，用于研究远程决定因素对配体结合事件的影响，反过来，配体结合信号向邻近结构元素的传播。因此，这些研究的相关性远远超出了EF-Hand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.