Binding Mechanisms and Conformational Equilibria in Biomacromolecular Interactions

生物大分子相互作用中的结合机制和构象平衡

• 批准号: RGPIN-2014-05776
• 负责人: Mittermaier, Anthony
• 金额: $ 3.93万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588609
• 关键词: Binding; Mechanisms; Conformational; Equilibria; Biomacromolecular

Understanding how biological macromolecules recognize and bind to their cognate ligands is essential for the rational design of drugs, biocatalysts, and for explaining biology at the atomic level. However, developing rigorous, quantitative descriptions of these processes is extremely challenging for a number of reasons. Firstly, biological macromolecules such as proteins and DNA are inherently flexible and populate ensembles of different conformations at equilibrium. In the presence of a cognate ligand, the relative populations of the conformers can be dramatically redistributed. This coupling between ligand binding and conformational changes can potentially make large contributions to the affinities of biomacromolecular interactions and can mediate allosteric communication between distant binding sites. However, it remains difficult to characterize in detail their conformational heterogeneity and harder still to quantify the contributions of structural and dynamical changes to the thermodynamics of binding. Secondly, the kinetics of molecular recognition events depend upon weakly-populated intermediate configurations of binding partners. In many cases the nature of the kinetic bottleneck is not well understood. For example, the access of ligands to deeply buried binding sites in proteins often requires some degree of conformational rearrangement of the protein in order to permit passage. However the extent of the structural distortions and the associated energetic costs are not well understood. The aim of this proposal is to gain a deeper understanding of biomacromolecular recognition at the atomic level by developing new experimental tools for characterizing binding events and applying them to model systems that exemplify the complexity of the phenomenon. Our experimental approach is based on combining high-field solution nuclear magnetic resonance (NMR) spectroscopy with calorimetry and mutagenesis. NMR spectroscopy is exquisitely sensitive to biomacromolecular structure and dynamics, making it a powerful tool for characterizing how conformational sampling is perturbed by ligand binding. Many NMR measurements can be interpreted quantitatively in terms of exchange rates or thermodynamic differences between conformational states, such as folded and unfolded or ligand-free and ligand-bound forms. In this regard, biological NMR data are highly complementary to those of isothermal titration calorimetry and differential scanning calorimetry. These methods directly quantify the thermodynamics of binding and folding reactions, respectively. Furthermore, mutagenesis permits the selective removal or introduction intra- and inter-molecular contacts. Using NMR, calorimetry, and mutagenesis, we will dissect binding pathways with atomic resolution in terms of specific chemical moieties on the macromolecule and ligand. In addition, we propose to develop new NMR approaches to characterize the structural and orientational heterogeneity of bound ligands. Ligand dynamics in the bound state can potentially have a significant impact on the free energy of binding, particularly on the entropic component, however it has typically been challenging to address experimentally. We will apply this combined approach to study ligand binding by guanine quadruplex DNA, a promising target in cancer therapeutics. We will determine the impact of coupled local folding and binding on DNA recognition by a homeodomain. We will elucidate how ligands gain access to a buried site in an odorant binding protein and characterize their dynamics once bound. This research program is unique world-wide in its approach and scope and will provide a detailed and rigorous new perspective on how biological macromolecules recognize and bind to their targets.

了解生物大分子如何识别和结合到它们的同源配体对于药物、生物催化剂的合理设计以及在原子水平上解释生物学是至关重要的。然而，由于多种原因，对这些过程进行严格的定量描述极具挑战性。首先，生物大分子如蛋白质和DNA具有固有的柔性，并且在平衡时具有不同构象的集合体。在同源配体的存在下，构象异构体的相对数量可以显著地重新分布。配体结合和构象变化之间的这种耦合可以潜在地对生物大分子相互作用的亲和力做出很大贡献，并且可以介导远距离结合位点之间的变构通信。然而，它仍然很难详细描述其构象的异质性和更难量化的结构和动力学变化的结合热力学的贡献。其次，分子识别事件的动力学依赖于结合伴侣的弱填充中间构型。在许多情况下，动力学瓶颈的性质并没有得到很好的理解。例如，配体进入蛋白质中深埋的结合位点通常需要蛋白质的某种程度的构象重排以允许通过。然而，结构扭曲的程度和相关的能量成本还没有得到很好的理解。该提案的目的是通过开发新的实验工具来表征结合事件并将其应用于模拟该现象的复杂性的模型系统，从而更深入地了解原子水平上的生物大分子识别。 我们的实验方法是基于结合高场溶液核磁共振（NMR）光谱与量热法和诱变。NMR光谱对生物大分子结构和动力学非常敏感，使其成为表征配体结合如何干扰构象采样的有力工具。许多NMR测量可以根据构象状态之间的交换率或热力学差异进行定量解释，例如折叠和未折叠或无配体和配体结合形式。在这方面，生物NMR数据与等温滴定量热法和差示扫描量热法的数据高度互补。这些方法分别直接量化结合和折叠反应的热力学。此外，诱变允许选择性去除或引入分子内和分子间接触。使用核磁共振，量热法，和诱变，我们将解剖结合途径与原子分辨率的大分子和配体上的特定化学部分。此外，我们建议开发新的NMR方法来表征结合配体的结构和取向的异质性。结合态中的配体动力学可能对结合的自由能，特别是对熵分量具有潜在的显著影响，然而，它通常具有挑战性，难以通过实验来解决。 我们将应用这种组合的方法来研究配体结合鸟嘌呤四链体DNA，一个有前途的目标，在癌症治疗。我们将确定耦合的局部折叠和结合的DNA识别的同源结构域的影响。我们将阐明配体如何获得一个埋藏的气味结合蛋白的网站，并描述其动态一旦绑定。该研究计划在其方法和范围方面是世界上独一无二的，将为生物大分子如何识别和结合其目标提供详细而严格的新视角。