The underlying dynamic exchange that dictates serine protease function

蛋白水解酶由一大群负责裂解(或加工)各种底物的酶组成。一类被称为丝氨酸蛋白酶的酶被预测在非活性构象和活性构象之间移动。这些运动是丝氨酸蛋白酶发挥作用的关键，也是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称为剥离毒素(ETA)的单一位置已经被确定，它可以将该酶完全移动到其失活或活性构象。通过对天然氨基酸的诱变来改变这个单一的位点，可以确定ETA的明确的非活性结构和活性结构。进一步的方法也将被用来理解ETA的不同部分如何将非活性构象和活性构象之间的构象变化传递到酶的不同部分，通常被称为“变构”。该项目将通过多个项目培训未被充分代表的群体的成员，包括多文化学生研究生体验(GEMS)和通过医学院的一个名为科罗拉多研究体验(CORE)的新项目。这项研究将利用新发现的单一内在动态残基，ETA D164，位于活跃部位附近。这种残基起着关键的“开关”的作用，控制着对丝氨酸蛋白酶十多年前提出的对非活性构象和活性构象的固有的全局采样。通过诱变，通过化学位移扰动(CSP)观察到溶液系综中的位移，这与两个方向中的任何一个方向的位移活性相关。因此，本研究的目标是：1)确定ETA中耦合的变构网络，它们调节非活性构象和活性构象的全局采样；2)确定非活性构象和活性构象采样的动力学和结构基础。这个项目得到了分子和细胞生物科学部分子生物物理学分部的支持。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.