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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611082
• 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，Alltooth Welle被称为血清溶质的载体（例如脂肪酸），抑制了展开的蛋白质和淀粉样蛋白肽的聚集（例如Aß）。分子分子分子对原型人血清白蛋白（HSA）的电流产生了重要的贡献。 Apploch G。通过位点的Aß低聚物是独特的，并且与低低的低分子量（MW）配体的相互作用是与Aß低聚物的成分。 HSA充其量是很少的。单一分辨率在长期​​内，将延伸到淀粉样蛋白酶的肽和其他细胞外伴侣。较低的配体的位点无法解决地点。其他NMR的进展中，其他配体包括我们率先绘制的方法，否则，我们将从隔离的HSA域进行分析，并将其分析以更长的方式扩展，否则，否则绘制了难以置信的变构网络。配体绑定，两个目标的gatientary数据可用性均以可用的材料和实验方法来实现，并利用OUBLICAT离子使用的NMR方法。分子分子分子分子mectra-核心伴侣伴侣以及配体与M与M的变构偶联提供独特的HQP训练机会。