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.

血浆的拥挤环境显著改变了控制血浆蛋白功能的动力学。因此，我们研究计划的长期目标是研究血浆蛋白在其天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。使用核磁共振（NMR）光谱和互补技术（例如荧光和电子显微镜）的多学科组合，我的小组作出了重大贡献，目前的理解如何血浆April，聚集倾向的内在无序多肽（IDP），与血浆分子伴侣，如人血清白蛋白（HSA）和表没食子儿茶素没食子酸酯相互作用。然而，到目前为止，我们的研究仅限于缓冲溶液，不能概括生理血浆条件的全部复杂性。初步的数据表明，血浆环境控制动态过渡从扩展到紧凑的构象，通过空间排斥和弱结合蛋白质溶质的组合作用的假设。因此，预期血浆会改变球状蛋白的解折叠热力学和IDP可接近的构象集合，这决定了对突变和翻译后修饰的变构反应。尽管如此，目前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的知识还很少。因此，我们的第一个短期目标是：（i）绘制α-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-突触核蛋白是释放到血浆中作为细胞间通讯介质的代表性IDP。这将是第一次在生理血浆条件下绘制IDP的变构网络。为了补充目标（i），第二个短期目标将集中在球状血浆蛋白，即我们计划：（ii）映射HSA与血浆环境的相互作用，并确定它们如何调节HSA展开。这一目标将揭示哪些HSA位点与哪些血浆拥挤物相互作用，以及这些相互作用如何调节HSA稳定性。初步数据表明，这两个目标是可行的，在现有的材料和实验方法，利用我们过去的出版物中采用的NMR方法。从长远来看，我们将把我们的血浆核磁共振计划扩展到其他典型的细胞外IDP和球状蛋白，以破译血浆蛋白在天然条件下如何发挥作用的一般可转移规则，以弥合体外与体内的差距并设计新的血液分析和血液分馏技术。因此，我们预计将产生广泛的影响，这将是科学和教育，因为拟议的计划提供了独特的培训机会，我们多样化的HQP队列。