Binding Mechanisms and Conformational Equilibria in Biomacromolecular Interactions

了解生物大分子如何识别和结合它们的同源配体，对于合理设计药物、生物催化剂以及在原子水平上解释生物学是至关重要的。然而，由于许多原因，开发对这些过程的严格、定量的描述是非常具有挑战性的。首先，蛋白质和DNA等生物大分子具有内在的灵活性，在平衡状态下聚集着不同构象的系综。在同源配体的存在下，构象分子的相对种群可以显著地重新分布。这种配体结合和构象变化之间的耦合可能对生物大分子相互作用的亲和力做出巨大贡献，并可以介导远距离结合位点之间的变构通讯。然而，仍然很难详细地表征它们的构象异质性，更难量化结构和动力学变化对结合热力学的贡献。其次，分子识别事件的动力学依赖于结合伙伴的弱填充中间构型。在许多情况下，动力瓶颈的性质没有得到很好的理解。例如，配体访问深埋在蛋白质中的结合部位通常需要蛋白质进行一定程度的构象重排才能通过。然而，结构扭曲的程度和相关的能源成本并没有得到很好的理解。这项建议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展示该现象复杂性的模型系统，从而在原子水平上更深入地了解生物大分子识别。**我们的实验方法基于将高场溶液核磁共振(NMR)光谱与量热和诱变相结合。核磁共振波谱对生物大分子结构和动力学非常敏感，使其成为表征构象采样如何受到配体结合干扰的有力工具。许多核磁共振测量可以根据交换率或构象状态之间的热力学差异来定量解释，例如折叠和展开或无配体和配体结合的形式。在这方面，生物核磁共振数据与等温滴定量热法和差示扫描量热法的数据具有很强的互补性。这些方法分别直接量化了结合反应和折叠反应的热力学。此外，诱变允许选择性地移除或引入分子内和分子间的接触。利用核磁共振、量热法和诱变，我们将根据大分子和配体上的特定化学部分，剖析具有原子分辨率的结合途径。此外，我们建议开发新的核磁共振方法来表征结合配体的结构和取向的异质性。结合态下的配基动力学可能对结合自由能有很大的影响，尤其是对结合的熵分量，然而这通常在实验上是有挑战性的。**我们将应用这种结合的方法来研究鸟嘌呤四链DNA的配基结合，这是癌症治疗中的一个有前途的靶点。我们将确定耦合的局部折叠和结合对同源结构域识别DNA的影响。我们将阐明配体如何进入气味结合蛋白的掩埋位置，并在结合后表征其动力学。这一研究计划在方法和范围上是世界上独一无二的，并将为生物大分子如何识别和结合其目标提供详细和严谨的新视角。

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.