Parvalbumin (PV) is a calcium binding protein expressed in humans, fish and avian species. In these organisms, the calcium (Ca²⁺) affinities of specific PV isoforms can vary by orders of magnitude. Despite the availability of high resolution structural data for many PV isoforms, the structural bases for how such proteins confer widely-varying divalent Ca²⁺ affinities and selectivities against common ions like magnesium (Mg²⁺) has been difficult to rationalize. We therefore conducted molecular simulations of several α-pavalbumin (α-parvalbumin (αPV)) constructs with Ca²⁺ affinities in the micromolar to nanomolar ranges to identify properties of conformations that contribute to their wide-ranging binding constants and selectivities against Mg²⁺. Specifically we examined D94S/G98E construct with a reported lower Ca²⁺ affinity (≈ -18.2 kcal/mol) relative to the WT (≈-22 kcal/mol), an S55D/E59D variant with enhanced affinity (≈-24 kcal/mol), and a truncated variant of αPV with weak affinity (≈-12.6 kcal/mol). We performed molecular dynamics simulations of these constructs and assessed their Ca²⁺ and Mg²⁺ binding properties using scores from molecular mechanics generalized Born approximation (MM/GBSA), ion/oxygen coordination patterns and thermodynamics via mean spherical approximation (MSA) theory, as well as via metrics of protein structure and hydration. Our key findings are that although MM/GBSA and MSA scores successfully rank-ordered the variants according to their previously-published affinities and Mg²⁺ selectivity, importantly, properties of Ca²⁺ loops in CBPs such as coordination, and charge are alone insufficient to rationalize their binding properties. Rather, Ca²⁺ affinity and selectivity against ²⁺ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions from protein folding and solubility. Our findings broaden our understanding of the molecular bases governing αPV ion binding that are likely shared by many Ca²⁺ binding proteins.

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EF手结合位点以外的钙结合的结构决定因素：α小白蛋白的研究

小白蛋白（PV）是在人类，鱼类和鸟类中表达的钙结合蛋白。在这些生物中，特定PV同工型的钙（Ca ²⁺）亲和力可以变化几个数量级。尽管可以获得许多PV异构体的高分辨率结构数据，但很难合理化此类蛋白质如何赋予广泛的二价Ca ²⁺亲和力和针对常见离子（如镁（Mg ²⁺））的选择性的结构基础。因此，我们对几种带有Ca ^2+的α-小白蛋白（α-小白蛋白（αPV））进行了分子模拟在微摩尔至纳摩尔范围内的亲和力，以鉴定构型的性质，这些构型有助于其广泛的结合常数和对Mg ^2+的选择性。具体来说，我们检查了D94S / G98E构建体，其报道的Ca ²⁺亲和力相对于WT（≈-22kcal / mol）较低，而WT（≈-22kcal / mol）具有增强的亲和力（≈-24kcal /摩尔）和亲和力较弱的αPV截短变体（≈-12.6kcal / mol）。我们对这些结构进行了分子动力学模拟，并评估了它们的Ca ²⁺和Mg ²⁺使用分子力学广义Born近似（MM / GBSA），离子/氧配位模式和热力学的分数通过平均球面近似（MSA）理论以及蛋白质结构和水合度进行评分。我们的主要发现是，尽管MM / GBSA和MSA分数已根据它们先前发布的亲和力和Mg ²⁺选择性成功地对变体进行了排序，但重要的是，单独的CBP中Ca ²⁺环的性质（如配位和电荷）不足使其结合特性合理化。相反，Ca ^2+的亲和力和对^2+的选择性是蛋白质的离子结合位点内的局部效应以及蛋白质折叠和溶解度的非局部贡献所产生的新兴特性。我们的发现拓宽了我们对控制αPV离子结合的分子碱基的理解，这些分子碱基可能与许多Ca ²⁺结合蛋白共有。