In Situ NMR Studies of Plasma Protein Dynamics and Interactions

血浆蛋白质动力学和相互作用的原位核磁共振研究

• 批准号: RGPIN-2019-05990
• 负责人: Melacini, Giuseppe
• 金额: $ 5.97万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748250
• 关键词: Situ; NMR; Studies; Plasma; Protein

The crowded environment of blood plasma significantly alters the dynamics that control the function of plasma proteins. Hence, the long-term goal of our research program is  to investigate plasma proteins in their native physiological environment and elucidate the in situ molecular mechanisms underlying how protein-plasma interactions modulate functional dynamics and allostery. Using a multidisciplinary combination of nuclear magnetic resonance (NMR) spectroscopy and complementary techniques (e.g. fluorescence and electron microscopy), my group has made significant contributions to the current understanding of how plasma Aß, an aggregation-prone intrinsically disordered polypeptide (IDP), interacts with plasma chaperones, such as human serum albumin (HSA) and epigallocatechin gallate. However, our studies have so far been limited to buffered solutions that do not recapitulate the full complexity of physiological plasma conditions. Preliminary data suggest the hypothesis that the plasma environment controls dynamic transitions from extended to compact conformations through the combined action of steric exclusion and weak binding to protein solutes. As a result, plasma is expected to alter the unfolding thermodynamics of globular proteins and the conformational ensembles accessible to IDPs, which dictate the allosteric response to mutations and post-translational modifications. Nonetheless, only scant knowledge is currently available on how plasma proteins interact with the rest of the plasma environment and on how these interactions affect allostery and unfolding. Hence, our first short-term objective is: (i) to map the interactions and allosteric networks of a-synuclein in plasma and in crowded solutions. a-synuclein is a representative IDP released into plasma as a mediator of cell-to-cell communication. This will be the first time that the allosteric networks of an IDP are mapped under physiological plasma conditions. To complement aim (i), the second short-term objective will focus on a globular plasma protein, i.e. we plan: (ii) to map the interactions of HSA with the plasma environment and determine how they modulate HSA unfolding. This aim will reveal which HSA sites interact with which plasma crowders and how these interactions modulate HSA stability. Preliminary data indicate that both aims are feasible in terms of available materials and experimental methods, capitalizing on NMR approaches employed in our past publications. In the long term, we will extend our in plasma NMR program to other prototypical extracellular IDPs and globular proteins to decipher general transferable rules for how plasma proteins function under native conditions, as needed to bridge the in vitro vs. in vivo gap and design new blood-based assays and blood fractionation technologies. Hence, we expect a broad impact, which will be both scientific and educational, as the proposed program provides unique training opportunities for our diverse HQP cohort.

血浆拥挤的环境显著改变了控制血浆蛋白质功能的动态。因此，我们研究计划的长期目标是研究血浆蛋白质在其自然生理环境中的作用，并阐明蛋白质-血浆相互作用如何调节功能动力学和变构的原位分子机制。利用核磁共振波谱和互补技术(例如荧光和电子显微镜)的多学科组合，我的团队为当前了解血浆Aü(一种易于聚集的固有无序多肽)如何与血浆伴侣蛋白相互作用做出了重大贡献，如人血清白蛋白(HSA)和表没食子儿茶素没食子酸酯。然而，到目前为止，我们的研究仅限于缓冲溶液，不能概括生理性等离子体条件的全部复杂性。初步数据表明，等离子体环境通过空间排斥和与蛋白质溶质的弱结合共同作用，控制着从扩展构象到紧凑构象的动态转变。因此，血浆有望改变球形蛋白质的展开热力学和IDPs可获得的构象系综，这决定了变构对突变和翻译后修饰的反应。然而，关于血浆蛋白如何与血浆环境的其余部分相互作用，以及这些相互作用如何影响变构和展开，目前还知之甚少。因此，我们的第一个短期目标是：(I)绘制a-突触核蛋白在血浆和拥挤溶液中的相互作用和变构网络。α-突触核蛋白是一种具有代表性的IDP，作为细胞间通讯的媒介释放到血浆中。这将是第一次在生理等离子体条件下映射IDP的变构网络。为了补充AIM(I)，第二个短期目标将集中在球状血浆蛋白上，即我们计划：(Ii)绘制HSA与血浆环境的相互作用图，并确定它们如何调节HSA的展开。这一目标将揭示哪些HSA位点与哪些血浆聚集物相互作用，以及这些相互作用如何调节HSA的稳定性。初步数据表明，这两个目标在可用材料和实验方法方面都是可行的，利用了我们过去出版物中采用的核磁共振方法。从长远来看，我们将把我们的血浆内核磁共振计划扩展到其他典型的胞外IDPs和球状蛋白质，以破译血浆蛋白质在自然条件下如何发挥作用的一般可转移规则，以弥补体外和体内的差距，并设计新的基于血液的分析和血液分离技术。因此，我们预计会产生广泛的影响，既是科学的，也是教育的，因为拟议的计划为我们不同的HQP队列提供了独特的培训机会。