Solution NMR Studies of Interactions of Ligands With Plasma Proteins

配体与血浆蛋白相互作用的溶液核磁共振研究

• 批准号: RGPIN-2014-04514
• 负责人: Melacini, Giuseppe
• 金额: $ 5.17万
• 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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=628840
• 关键词: Solution; NMR; Studies; Interactions; Ligands

Plasma proteins, although well known as carriers of serum solutes (e.g. fatty acids), have been recently shown to function also as extra-cellular chaperones, which inhibit the aggregation of unfolded proteins and amyloidogenic peptides (e.g. Aß). The long-term goal of our research program is to define fundamental molecular mechanisms governing ligand binding and aggregation inhibition by plasma proteins. My laboratory has made significant contributions to the current understanding of how the prototypical human serum albumin (HSA) acts as the most potent inhibitor of Aß fibrillization in plasma. Using a multidisciplinary combination of nuclear magnetic resonance (NMR) spectroscopy and other biophysical and biochemical approaches (e.g. fluorescence, dynamic light scattering, electron microscopy and site-directed mutagenesis), we have shown that HSA prevents the Aß peptide from forming insoluble amyloid fibrils by selectively binding to soluble Aß oligomers and competing with the further addition of Aß monomers. We have determined the stoichiometry and the affinities of the HSA - Aß oligomer complexes and through comparative mutational analyses we have shown that HSA recognizes the Aß oligomers through sites that are unique and distinct from those interacting with low molecular weight (MW) ligands. Our preliminary data suggest the hypothesis that the critical contacts with the Aß oligomers are mediated by flexible loops of HSA with partial sequence homology to Aß. However, the current knowledge about the location of the Aß oligomer binding sites within HSA is at best scant. Our first short-term objective will therefore be to: (i) Map at single-residue resolution the binding sites within HSA for the Aß oligomers, by combining the multidisciplinary approach of our previous publications with new NMR experiments designed to probe reversible interactions with the Aß oligomers. This will be the first time that binding sites of a protein for oligomers of an amyloidogenic peptide are mapped at single-residue resolution. In the long term, this objective will be extended to a plethora of other amyloidogenic peptides and other extracellular chaperones. We will also elucidate the mechanisms underlying the other primary function of plasma proteins, i.e. ligand transport. The structures of ligand-bound HSA reveal the HSA architecture and the location of the binding sites for low MW ligands. However, structures alone cannot address several outstanding questions about ligand binding. How do ligands access buried binding sites? How does conformational entropy drive ligand binding? How are the apo conformational pre-equilibria allosterically coupled to ligand binding? To address these fundamental questions our second objective will be to: (ii) Comparatively analyze the dynamics of HSA in the absence and presence of fatty acids and other ligands, benefiting from recent NMR advances. These include an approach we pioneered to map otherwise elusive allosteric networks through the covariance analysis of NMR chemical shifts. We will start with the analysis of isolated HSA domains and will expand to progressively longer HSA constructs to reveal how dynamics modulates ligand binding, gating and allostery. The preliminary data available so far indicate that both objectives are feasible in terms of available materials and of experimental methods, capitalizing on the NMR approaches employed in our past publications. The resulting program will have a broad impact that goes well beyond HSA, as it will reveal general molecular mechanisms for the extra-cellular chaperones and for the allosteric coupling of ligand binding to protein dynamics. The impact will be both scientific and educational, as the proposed program provides a unique HQP training opportunity.

血浆蛋白虽然众所周知是血清溶质的载体（例如脂肪酸），但最近已证明其功能也充当细胞外链酮，抑制了展开的蛋白质和淀粉样蛋白植物肽的聚集（例如Aß）。我们的研究计划的长期目标是定义控制配体结合和血浆蛋白聚集抑制的基本分子机制。 My laboratory has made significant contributions to the Using a multidisciplinary combination of nuclear magnetic resonance (NMR) spectroscopy and other biophysical and biochemical approaches (e.g. fluorescence, dynamic light scattering, electronic microscopy and site-directed mutagenesis), we have shown that HSA prevents the Aß peppery from forming insoluble amyloid fibrils by Selectively binding to solid Aß低聚物并与进一步添加Aß单体竞争。我们已经确定了HSA -Aß低聚物复合物的化学计量和亲和力，并通过比较突变分析，我们表明，HSA通过与低分子量（MW）配体相互作用的位点识别Aß低聚物。我们的初步数据表明，与Aß低聚物的关键接触是由HSA的柔性循环介导的，其部分序列同源于Aß。但是，当前有关Aß低聚物结合位点位置的知识充其量很少。因此，我们的第一个短期目标将是：（i）通过将我们以前出版物的多学科方法与旨在证明与Aß寡聚物相反的相互作用的新的NMR实验相结合，将HSA内HSA内的绑定位点映射为Aß低聚物的结合位点。这将是首次将蛋白质的蛋白质的结合位点用于淀粉样蛋白肽的低聚物，以单递归分辨率映射。从长远来看，该目标将扩展到其他大量其他淀粉样蛋白生成肽和其他细胞外伴侣。我们还将阐明血浆蛋白的另一个主要功能的机制，即配体转运。配体结合HSA的结构揭示了HSA结构和低MW配体的结合位点的位置。但是，仅结构无法解决有关配体约束的几个杰出问题。配体如何访问构建的绑定站点？会议熵如何驱动配体结合？ Apo会议前均衡性如何与配体结合结合？为了解决这些基本问题，我们的第二个目标是：（ii）在不存在和存在脂肪酸和其他配体的情况下，相对分析了HSA的动态，从最近的NMR进步中受益。这些方法包括我们通过NMR化学移位的协方差分析来策划绘制原本难以捉摸的变构网络的方法。我们将从分析孤立的HSA域的分析开始，并将扩展到逐渐更长的HSA构建体，以揭示动力学如何调节配体结合，门控和变构。到目前为止，可用的初步数据表明，这两个目标在可用材料和实验方法方面都是可行的，并利用了我们过去出版物中所采取的NMR方法。最终的程序将产生广泛的影响，远远超出了HSA，因为它将揭示细胞外伴侣的一般分子机制以及配体结合与蛋白质动力学的变构耦合。拟议的计划提供了独特的HQP培训机会，因此影响既是科学又是教育意义。