Collaborative experimental & computational studies of conformational transitions

描述(由申请人提供)：蛋白质构象转变是信号传递、酶催化和细胞结构组装的基础。定量了解蛋白质如何在不同的折叠结构之间相互转换是生物学上的一项重大挑战；迎接这一挑战将对治疗大量与信号级联或酶有关的疾病产生影响。这项提议旨在通过描述蛋白质中的构象转变途径来了解控制蛋白质构象转变的物理原理。由于这些途径非常复杂，为了使这一目标可行，将对同源蛋白质进行比较。我们主要关注来自双组分系统家族(NTRC)的信号蛋白以及来自大肠杆菌和极端细菌的腺苷酸激酶(ADK)的同源物。这些ADK同系物在极端温度或压力下具有最佳的酶活性。提出了一种核磁共振动力学实验、先进计算方法和功能分析相结合的迭代方法。(1)实验测量了稳定构象态的结构和它们之间的相互转化速率，(2)原子细节地计算了过渡路径，(3)识别了促进途径的关键相互作用，(4)设计了使这些相互作用失效的突变，(5)通过实验测量了相互转化率的变化，(6)基于实验观察进行了新的计算。通过比较同源蛋白和突变体之间的转换途径，确定了导致机制差异的关键残基，并赋予了它们各自的温度或压力行为。此外，测定这些酶适应极端环境的相互转化熵和熵变化可能有助于揭示形成原始酶的进化选择机制。在更广泛的背景下，从分子途径获得的知识可能会阐明蛋白质构象转变的一般原理，从而将我们对蛋白质能量景观的理解从基态扩展到“过渡景观”。

DESCRIPTION (provided by applicant): Protein conformational transitions are fundamental to signaling, enzyme catalysis, and assembly of cellular structures. Developing a quantitative understanding of how proteins interconvert between different folded structures is a grand challenge in biology; meeting this challenge would have an impact in treating a large number of diseases that are linked to signaling cascades or enzymes. This proposal aims to understand the physical principles that control protein conformational transitions by characterizing transitio pathways in proteins. Since these pathways are very complex, homologous proteins will be compared to make this aim feasible. We focus on a signaling proteins from the two-component system family (NtrC) and homologs of the enzyme adenylate kinase (Adk) from E. coli and extremophiles. These Adk homologs have optimal enzymatic activity at extreme temperature or pressure. An iterative approach between NMR dynamics experiments, advanced computational methods and functional assays is proposed. (1) Structures of stable conformational states and rates of interconversion between them are measured experimentally, (2) transition pathways are computationally characterized in atomistic detail, (3) crucial interactions that facilitate pathway are identified, (4) mutations are designed that disable these interactions, (5) the resulting changes in interconversion rates are measured experimentally, and (6) new computations are performed based on experimental observations. By comparing transition pathways among homologous proteins and mutants key residues are identified that lead to mechanistic differences, and confer their respective temperature or pressure behaviors. Furthermore, determining interconversion entropy and entropy changes for these enzymes adapted to extreme environments may shed light on the evolutionary selection mechanisms that shaped primitive enzymes. In a broader context, knowledge gained from the molecular pathways may elucidate general principles of conformational transitions in proteins, thereby expanding our understanding of protein energy landscapes from the ground states to "transition landscapes".