Impact of the Unliganded State on Parvalbumin Divalent Ion Affinity

Parvalbumins是小的Ca 2+结合蛋白质，以其细胞溶质钙缓冲活性而闻名。小清蛋白（PV）家族的B分支表现出一系列的二价离子结合亲和力，鸟类胸腺激素（ATH）和大鼠b-PV分别处于高亲和力和低亲和力的极端，鸟类小清蛋白3（PV 3）处于中间。该项目的目标是阐明这些不同的金属离子结合特征的结构基础。现有的数据表明，离子亲和力是由金属离子结合基序本身以外的决定因素的影响;分子的N-末端AB区域和CD-EF金属离子结合域之间的相互作用可以调节离子亲和力;在unliganded蛋白质的结构差异是负责在一定程度上观察到的亲和力变化。本项目有四个具体目标：1）鉴定PV 3中相对于ATH减弱二价离子亲和力的氨基酸残基; 2）鉴定大鼠B中相对于PV 3减弱金属离子亲和力的那些残基; 3）获得大鼠a的无钙形式的高分辨率结构数据（高亲和力同种型）、大鼠B（低亲和力同种型）、鸡PV 3，以及时间允许的，由大鼠a和大鼠B的AB和CD-EF结构域形成的嵌合PV;和4）比较和对比这同一组蛋白质中的骨架动力学。这项研究有望为蛋白质-配体相互作用提供实质性的见解，说明蛋白质的非配体形式如何塑造配体结合的能量学。研究结果将与蛋白质设计与工程的新兴领域相关，此计画将提供有价值的训练给研究生与本科生。参与这项工作的个人将接触到蛋白质-配体相互作用的严格热力学分析和高分辨率蛋白质结构分析的尖端方法。这些数据将被整合到PI每年提供的基于问题的研究生物理生物化学课程中。这个不断发展的课程旨在为具有平均数学和物理技能的学生提供生物分子表征的物理方法的可访问介绍;为这些学生提供对这些物理工具的力量的欣赏;鼓励他们考虑将这些工具纳入自己的研究的方法，理想情况下，考虑生物物理学的职业生涯。

Parvalbumins are small Ca2+-binding proteins best known for their cytosolic calcium-buffering activity. The b branch of the parvalbumin (PV) family exhibits a spectrum of divalent ion-binding affinity, with avian thymic hormone (ATH) and rat b-PV at the high- and low-affinity extremes, respectively, and avian parvalbumin 3 (PV3) in the middle. The goal of this project is to elucidate the structural basis for these differing metal ion-binding signatures. Existing data suggest that ion affinity is influenced by determinants outside the metal ion-binding motifs per se; that the interaction between the N-terminal AB region of the molecule and the CD-EF metal ion-binding domain can modulate ion affinity; and that structural differences in the unliganded proteins are responsible in part for the observed variations in affinity. This project has four specific objectives: 1) to identify the amino acid residues in PV3 that attenuate divalent ion affinity relative to ATH; 2) to identify those residues in rat b that attenuate metal ion affinity relative to PV3; 3) to obtain high-resolution structural data on the Ca2+-free forms of rat a (a high-affinity isoform), rat b (a low-affinity isoform), chicken PV3, and, time permitting, chimeric PVs formed from the AB and CD-EF domains of rat a and rat b; and 4) to compare and contrast the backbone dynamics in this same set of proteins. This research is expected to provide substantial insight into protein-ligand interactions, illustrating how the unliganded form of the protein can shape the energetics of ligand binding. The results should have relevance to the nascent field of protein design and engineering.This project will provide valuable training to graduate and undergraduate students. Individuals involved with this effort will receive exposure to rigorous thermodynamic analysis of protein-ligand interactions and cutting-edge methods for high-resolution protein structure analysis. The data will be integrated into a problem-based graduate physical biochemistry course offered annually by the PI. This ever-evolving course seeks to provide an accessible introduction to physical methods of biomolecular characterization to students with average math and physics skills; to provide these students with an appreciation of the power of these physical tools; to encourage them to consider ways to incorporate these tools into their own research and, ideally, to consider a career in biophysics.