Dynamics and Thermal Stability in CFTR Function and Dysfunction

项目总结（见说明）： CFTR在对肺健康至关重要的离子/流体稳态中起着至关重要的调节作用，影响阴离子（直接）和阳离子（间接）转运。CFTR功能的复杂性需要一个复杂的和动态的结构，最近已经演变为一个独特的ABC转运蛋白家族成员。像所有蛋白质一样，CFTR进化必须在结构复杂性和热力学稳定性之间实现平衡，结果只有约25%的合成野生型多肽链达到稳定状态。 AF 508突变加剧了低效的生物合成，将这一比例降低到基本为零。包括降低温度、下调质量控制组分和所谓的“校正剂”小分子在内的一些操作改善了细胞加工和运输。然而，这些机动不能恢复热稳定性（TS），并且在温度<30 oC时的部分通道功能在37 oC时迅速丧失。 因此，有效的治疗需要恢复TS的试剂。在这里，我们专注于变构耦合途径，决定TS了解其机制的基础，使它可以被合理地操纵。SA 1将阐明NBD 1中调节插入（RI）的不稳定影响以及其切除如何恢复稳定性和功能。SA 2将采用两种互补的方法来鉴定恢复TS的其他变化，第一种利用分子动力学，第二种探索AF 508 CFTR保留TS的基础 其他物种的稳定性。这些方法已经揭示了在偶联途径中的关键位置处引入脯氨酸残基使AF 508 CFTR稳定。SA 3将利用CFTR的小分子和大分子粘合剂作为热稳定剂。首先，将通过计算筛选小分子文库（实验验证）以结合TS中涉及的蛋白质区域。其次，我们将鉴定更大的蛋白质/肽结合剂，包括合成抗体和纳米抗体。已经与这两个领域的主要实验室合作开发了有效的初始试剂。除了提供一个全新的范例与集成的计算和实验方法的CF问题，我们提供了这个PPG的其他项目与必要的CFTR工具和方法。

PROJECT SUMMARY (See instructions): The CFTR plays a crucial regulatory role in ion/fluid homeostasis essential to lung health, impacting both anion (directly) and cation (indirectly) transport. The complexity of CFTR function requires a complex and dynamic structure that has evolved relatively recently as a unique ABC transporter family member. Like all proteins, CFTR evolution had to achieve a balance between structural complexity and thermodynamic stability with the result that only ~25% of wild-type polypeptide chains synthesized achieve a stable state. The AF508 mutation exacerbates inefficient biogenesis, reducing this proportion to essentially zero. Some manipulations including reduced temperature, down-regulation of quality control components and so-called "corrector" small molecules improve cellular processing and trafficking. However, these maneuvers do not restore Thermal Stability (TS) and the partial channel function at temperatures < 30oC is rapidly lost at 37oC. Thus effective therapies require reagents that restore TS. Here we focus on the allosteric coupling pathways that determine TS to understand its mechanistic basis so that it can be manipulated rationally. SA 1 will elucidate the destabilizing influence of the Regulatory Insertion (Rl) in NBD1 and how its excision restores stability and function. SA 2 will employ two complementary approaches to identify other changes that restoreTS, the first utilizing molecular dynamics and the second exploring the basis of retained TS by AF508 CFTRs of some other species that are stable. These methods have already revealed that the introduction of proline residues at key positions in coupling pathways stabilize AF508 CFTR. SA 3 will utilize both small and larger molecule binders of CFTR as thermal stabilizers. First, small molecule libraries will be screened computationally (validated experimentally) for binding to regions of the protein implicated in TS. Second, we will identify larger protein/peptide binders including synthetic antibodies and nanobodies. Effective initial reagents have already been developed in collaboration with leading laboratories in both fields. In addition to providing an entirely new paradigm with integrated computational and experimental approaches to the CF problem, we provide the other projects of this PPG with essential CFTR tools and methodologies.