The underlying dynamic exchange that dictates serine protease function

蛋白酶包括负责切割（或加工）多种底物的一大组酶。一类蛋白酶，称为丝氨酸蛋白酶，被预测在非活性和活性构象之间移动。 这些运动对于丝氨酸蛋白酶的功能至关重要，并且代表了为什么酶的运动能力对其功能如此重要的最明显的例子之一。 在这项研究中，丝氨酸蛋白酶中的一个单一位点被称为剥脱毒素（ETA），它将这种酶完全移动到其非活性或活性构象。 通过诱变天然氨基酸改变这个单一位点，可以确定ETA的明确的无活性和活性结构。还将使用其他方法来理解ETA的不同部分如何将非活性构象和活性构象之间的这种构象变化传递给酶的不同部分，通常称为“变构”。该项目将通过多个项目培训代表性不足的群体的成员，包括多元文化学生的研究生体验（GEMS）和一个新的计划，通过医学院称为科罗拉多研究经验（CORE）。这项研究将利用一个单一的内在动态残留物，ETA D164，位于活性位点附近的新鉴定。这个残基作为一个关键的“开关”，控制固有的全局采样的非活性和活性构象，提出了丝氨酸蛋白酶在十多年前。通过诱变，观察到溶液系综中的位移，如通过化学位移扰动（CSP）在两个方向中的任一个上监测的，其与在两个方向中的任一个上位移的活性相关。因此，本研究的目标是：1）识别ETA内耦合的变构网络，其调节非活性和活性构象的全局采样; 2）确定非活性和活性构象的这种采样的动力学和结构基础。该项目由分子和细胞生物科学部的分子生物物理学小组支持。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Proteases comprise a large group of enzymes responsible for cleaving (or processing) a wide array of substrates. One class of proteases, called serine proteases, are predicted to move between inactive and active conformations. These movements are critical for serine proteases to function and represent one of the clearest examples of why the ability of an enzyme to move is so important for their functions. In this research, a single site within the serine protease called exfoliative toxin (ETA) has been identified that moves this enzyme either completely to its inactive or active conformation. By changing this single site through mutagenesis of the natural amino acid, the explicit inactive and active structures of ETA can be determined. Further methods will also be used to understand how different parts of ETA communicate this conformational change between inactive and active conformations to different parts of the enzyme, referred to generally as “allostery”. This project will train members of the underrepresented groups through multiple programs, including the Graduate Experience for Multicultural Students (GEMS) and a new program through the School of Medicine called Colorado Research Experiences (CORE).This research will capitalize on the novel identification of a single inherently dynamic residue, ETA D164, situated adjacent to the active site. This residue acts as a pivotal “switch” that governs the inherent global sampling of inactive and active conformations, as proposed for serine proteases over a decade ago. Through mutagenesis, shifts in solution ensembles are observed as monitored via chemical shift perturbations (CSPs) in either of two directions which correlates to activity to shift in either of two directions. Thus, the goals in this research are to 1) Identify the allosteric networks coupled within ETA that modulate global sampling of inactive and active conformations and 2) determine both the dynamic and structural basis of this sampling of inactive and active conformations. This project is supported by the Molecular Biophysics Cluster of the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.