Functional Dynamics and Activation Mechanisms in Enzymes

酶的样品状态准备好结合底物，形成米氏中间体，采用促进化学步骤的构象，并获得能够释放产品的状态，同时最大限度地减少反向过程。催化是通过功能状态的动态平衡实现的，称为系综。核磁共振超越了结晶学，除了能够实现催化的快(局部)和慢(合作)动力学之外，还捕捉到了基态和激发态的高分辨率结构。系综视角对于推进我们对蛋白质功能的理解是极其重要的，也是极具挑战性的，因为我们试图沿着整个反应坐标路径描述所有具有原子分辨率的构象。虽然可以研究许多酶，但我们将重点放在氟乙酸酯脱卤酶(FAcD)作为一个模型酶上，因为它是一个同源二聚体(蛋白质中最常见的基序)，因此引发了关于分子内和分子间异构性的问题，分子内和分子间异构性是蛋白质功能的核心，也是最不被了解的。FAcD还可以解毒一种含氟毒物，代表了一个有趣的模型来追求突变、功能进化和生物修复。我们将解决：1.功能状态的动态平衡。我们将确定关键的功能态，确定系综自由能图景，重点放在州际动力学上，并解决至少一个激发态(准备进行衬底捕获)的结构，该结构没有晶体结构。2.变构网络与机理。甲基(TROSY)和19F核磁共振将提供一个跨越二聚体的化学位移网络。化学位移作为抑制剂、底物类似物和D2O的函数的协方差分析将产生变构网络，并解决氢键水作为变构开关的关键作用。3.酶动力学与催化。将通过主链和侧链松弛实验评估整个二聚体的动力学。我们将研究催化效率和分子动力学之间的联系。4.底物抑制。我们将研究二聚体中的变构底物口袋在高浓度时如何起到抑制作用，而在低浓度时如何起到有效的催化作用。5.~(19)F-核磁共振指定结晶学。我们将改进DFT和局部MD模拟的使用，作为19F核磁共振任务中诱变的替代方法。6.二聚体的蛋白质折叠。变性剂和压力将被用来研究这种具有原子分辨率的二聚体的复杂折叠过程。7.蛋白质的诱变与进化。将通过单细胞(FACS)方法制造和研究大量的变异体。将测试变构网络&酶将朝着更高的催化效率或底物混杂的方向进化。结果将极大地促进从系综的角度理解结构和动力学在催化中的作用，同时解决关于原型二聚体中变构和水网络的几个范式转换问题。

Enzymes sample states primed to bind substrates, form Michaelis intermediates, adopt conformers facilitating chemical steps, & acquire states enabling product release, while minimizing the reverse process. Catalysis is enabled by a dynamic equilibrium of functional states, referred to as the ensemble. NMR goes beyond crystallography to capture high-resolution structures of ground & excited states in addition to both fast (local) & slow (cooperative) dynamics enabling catalysis. The ensemble perspective is both profoundly important to advancing our understanding of protein function & profoundly challenging, since we seek to describe all conformers with atomic resolution along the entire reaction coordinate pathway. While many enzymes could be studied, we focus on fluoroacetate dehalogenase (FAcD) as a model enzyme namely because it is a homodimer (the most common motif among proteins) thus inviting questions regarding intra- and intermolecular allostery, which is at the heart of protein function & least understood.  FacD also detoxifies a fluorinated poison & represents an interesting model to pursue mutagenesis, functional evolution, & bioremediation.  We will address: 1. The dynamic equilibrium of functional states. We will identify key functional states, determine the ensemble free energy landscape with a focus on interstate dynamics & solve a structure of at least one excited state (poised for substrate capture) for which there is no crystal structure. 2. Allosteric networks & mechanisms. Methyl (TROSY) & 19F NMR will provide a network of chemical shifts across the dimer. Covariance analysis of chemical shifts as a function of inhibitor, substrate analogue, & D2O will generate an allosteric network & address the critical role of hydrogen-bonded waters as allosteric switches. 3. Enzyme dynamics & catalysis. Dynamics will be assessed across the dimer via backbone & side chain relaxation experiments. We will study the connection between catalytic efficiency & molecular dynamics. 4. Substrate inhibition. We will study how an allosteric substrate pocket in the dimer contributes to inhibition at high concentrations & efficient catalysis at low concentrations. 5. 19F NMR assignments crystallography. We will improve the use of DFT & local MD simulations as an alternative to mutagenesis for 19F NMR assignments. 6. Protein folding of a dimer. Both denaturant & pressure will be used to study the complex folding process of this dimer with atomic resolution. 7. Protein mutagenesis & evolution. High numbers of variants will be made & studied via single cell (FACS) methods. Allosteric networks will be tested & the enzyme will be evolved toward greater catalytic efficiency or substrate promiscuity. Results will greatly advance understanding of the role of structure & dynamics in catalysis from the perspective of an ensemble, while addressing several paradigm shifting questions regarding allostery & water networks in a prototypical dimer.