Alpha and Beta Parvalbumins: Functional Consequences of Divergent Tertiary Interactions

9603877 Henzl尽管它们一般相似，但小清蛋白（PV）同种型表现出实质性的物理和功能多样性。 该项目将研究实现异构体依赖性变异的三种潜在机制。 三级之间的相互作用的C-末端和残基26- 36的重要性将在选择特定位点的PV变体-采用差示扫描量热法，Ca 2+结合测量，滴定量热法，和15 N NMR弛豫测量检查。 为了评估总体静电自由能对金属离子结合行为的影响，将在等电点差异很大的PV亚型中检查S55 D和G98 D突变对Ca 2+和Mg 2+结合亲和力的影响。 最后，将检查金属离子促进的CPV 3（禽PV）缔合的最小序列要求。 络合的能量学将通过分析超离心和扫描量热法来研究。 三聚体的低分辨率结构信息将通过抗体反应性和化学交联研究获得。 将通过X射线晶体学和/或NMR获得详细的结构数据。 当代蛋白质研究试图确定决定蛋白质结构和功能的物理原理。 小白蛋白为研究这些问题提供了一个有吸引力的系统。 脊椎动物特异性的小Ca 2+结合蛋白家族说明了相同的基本蛋白质结构在解决不同的生物学问题中的应用-PV在维持细胞溶质Ca 2+水平低于毒性阈值方面发挥相当被动的作用。 相比之下，在高等脊椎动物中，-PV用于高度专门化的信号作用。 这种功能变化伴随着物理性质的相应变化。 例如，-PV显示的结构刚度大于-PV。 因此，本项目的一个目标是研究这种差异的分子基础。 此外，尽管所有的-PV都非常紧密地结合Ca 2+，但-PV显示出对离子的高度可变的亲和力。 因此，本项目的第二个目标是评估总电荷对金属离子结合性能的影响。 最后，尽管小清蛋白被广泛认为是非缔合单体蛋白，但禽-PV CPV 3在Ca 2+或Mg 2+存在下形成三聚体复合物。 因此，本项目的第三个目标是确定负责这种自我互补的结构特质。这项工作将阐明通过细微的序列改变来定制大分子的物理稳定性和配体结合特性的明显成功的策略。 从这些研究中获得的知识可能有助于实现操纵现有生物活动和设计新活动的长期目标。

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.