The underlying dynamic exchange that dictates serine protease function

决定丝氨酸蛋白酶功能的潜在动态交换

• 批准号: 2332239
• 负责人: Elan Eisenmesser
• 金额: $ 68.76万
• 依托单位: University of Colorado at Denver
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2332239&HistoricalAwards=false
• 关键词: underlying; dynamic; exchange; dictates; serine

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.

蛋白酶包括一大批负责清洁（或加工）各种底物的酶。预测一类称为丝氨酸蛋白的蛋白酶在无活性和活性构象之间移动。这些运动对于丝氨酸蛋白的功能和代表了酶移动能力对其功能如此重要的原因的最清晰的例子之一至关重要。在这项研究中，已经鉴定出称为脱落性毒素（ETA）的丝氨酸蛋白中的单个位点，该蛋白被确定为将该酶完全移至其无活性或活性构象。通过通过天然氨基酸的诱变改变这一位点，可以确定ETA的显式无活性和活性结构。进一步的方法还将用于了解ETA的不同部分如何将这种构象变化与酶的不同部分之间的这种构象变化传达，通常称为“变构”。该项目将通过多个计划培训代表性不足的小组的成员，包括用于多元文化学生的研究生体验（GEMS）和通过名为Colorado Research Experience（Core）的医学院的新计划（CORE）。这项研究将利用新的固有动态住所ETA D164的新颖识别，即ETA D164，位于活动现场附近。该居住地充当了一个关键的“开关”，它控制着十年前针对丝氨酸蛋白所提出的无活性和活性构象的全球抽样。通过诱变，通过化学移动扰动（CSP）在两个方向中的任何一个中观察到溶液集合的变化，这将与活性与两个方向中的任何一个相关。这是这项研究的目标是1）确定ETA中耦合的变构网络调节无效和主动构象的全局采样，2）确定这种无效和主动构象采样的动态和结构基础。该项目得到了分子和细胞生物科学划分的分子生物物理学群集的支持。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响审查标准，通过评估诚实地认为支持了支持。