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.

血浆的拥挤环境显着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，并阐明蛋白质如何作用的原位分子机制。等离子体相互作用调节功能动力学和变构，我的团队利用核磁共振（NMR）光谱和互补技术（例如荧光和电子显微镜）的多学科组合，为当前对等离子体如何发挥作用的理解做出了重大贡献。 Aß 是一种易于聚集的内在无序多肽 (IDP)，可与人血清白蛋白 (HSA) 和表没食子儿茶素没食子酸酯等血浆伴侣相互作用。初步数据表明，血浆环境通过空间排斥和与蛋白质溶质的弱结合的联合作用控制从延伸构象到紧凑构象的动态转变。因此，血浆有望改变球状蛋白的展开热力学和 IDP 易接近的构象集合，这决定了对突变和翻译后修饰的变构反应。然而，目前关于血浆蛋白如何相互作用的知识很少。因此，我们的第一个短期目标是：（i）绘制血浆和拥挤中 α-突触核蛋白的相互作用和变构网络。 α-突触核蛋白是释放到血浆中的代表性 IDP，作为细胞间通讯的介质，这将是首次在生理血浆条件下绘制 IDP 的变构网络，以补充目标 (i)。第二个短期目标将集中于球状血浆蛋白，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 展开。该目标将揭示哪些 HSA 位点与哪些位点相互作用。初步数据表明，利用我们过去出版物中使用的 NMR 方法，这两个目标在可用材料和实验方法方面都是可行的。与其他原型细胞外 IDP 和球状蛋白相结合，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，根据需要弥合体外与体内的差距，并设计新的基于血液的测定和血液分离技术。因此，我们期望产生广泛的影响，这将是科学和教育方面的，因为拟议的计划为我们多样化的 HQP 群体提供了独特的培训机会。