The underlying dynamic exchange that dictates serine protease function

蛋白酶包括一大类负责切割（或加工）各种底物的酶。有一类蛋白酶，称为丝氨酸蛋白酶，预计会在非活性构象和活性构象之间移动。这些运动对丝氨酸蛋白酶的功能至关重要，也是最清楚的例子之一，说明了为什么酶的运动能力对它们的功能如此重要。在这项研究中，已经确定了丝氨酸蛋白酶中称为剥脱毒素（ETA）的单个位点，该位点将该酶完全移动到非活性或活性构象。通过诱变天然氨基酸改变这一单一位点，可以确定ETA的显式非活性和活性结构。进一步的方法还将用于了解ETA的不同部分如何将这种非活性构象和活性构象之间的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）和医学院的一个名为科罗拉多研究体验（CORE）的新项目。本研究将利用对位于活性位点附近的单个固有动态残基ETA D164的新鉴定。这种残基作为一个关键的“开关”，控制着固有的非活性和活性构象的整体采样，就像十多年前提出的丝氨酸蛋白酶一样。通过诱变，通过化学位移扰动（csp）在两个方向中的任何一个方向上观察到溶液系的移动，这与在两个方向中的任何一个方向上移动的活性相关。因此，本研究的目标是：1)确定ETA内耦合的变构网络，该网络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采样的动态和结构基础。本项目由分子与细胞生物科学部分子生物物理组支持。该奖项反映了美国国家科学基金会的法定使命，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.