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。定量了解蛋白质如何在不同折叠结构之间相互转化是生物学中的一项巨大挑战。应对这一挑战将对治疗与信号级联或酶相关的大量疾病产生影响。 该提案旨在通过表征蛋白质中的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，因此将比较同源蛋白质以使这一目标变得可行。我们重点研究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微生物的腺苷酸激酶 (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。 提出了核磁共振动力学实验、先进计算方法和功能测定之间的迭代方法。 (1) 通过实验测量稳定构象状态的结构和它们之间的相互转化率，(2) 以原子细节计算表征转换途径，(3) 识别促进途径的关键相互作用，(4) 设计突变以禁用这些相互作用，(5) 通过实验测量所产生的相互转化率变化，以及 (6) 根据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变途径，鉴定出导致机制差异的关键残基，并赋予它们各自的温度或压力行为。此外，确定这些适应极端环境的酶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“转变景观”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".