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导、酶催化和细胞结构组装的基础。对蛋白质如何在不同折叠结构之间相互转化进行定量理解是生物学中的一大挑战；迎接这一挑战将对治疗大量与信号级联反应或酶有关的疾病产生影响。本提案旨在通过表征蛋白质中的过渡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，同源蛋白将进行比较，使这一目标可行。我们重点研究了来自大肠杆菌和极端微生物的双组分系统家族（NtrC）的信号蛋白和腺苷酸激酶（Adk）的同源物。这些Adk同源物在极端温度或压力下具有最佳的酶活性。提出了核磁共振动力学实验、先进计算方法和功能分析之间的迭代方法。(1)实验测量稳定构象态的结构和它们之间的相互转换速率，(2)在原子细节上计算表征跃迁途径，(3)确定促进途径的关键相互作用，(4)设计使这些相互作用失效的突变，(5)实验测量相互转换速率的变化，(6)基于实验观察进行新的计算。通过比较同源蛋白和突变体之间的转化途径，确定了导致机制差异的关键残基，并确定了各自的温度或压力行为。此外，确定这些酶适应极端环境的相互转化熵和熵的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，从分子途径中获得的知识可以阐明蛋白质构象转变的一般原理，从而扩大我们对蛋白质能量景观从基态到“过渡景观”的理解。

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".