RUI: Sortase-Mediated Ligations for Probing Binding Mechanisms of Large Disordered Proteins

RUI：分选酶介导的连接用于探测大无序蛋白质的结合机制

• 批准号: 2004237
• 负责人: John Antos
• 金额: $ 34.4万
• 依托单位: Western Washington University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2004237&HistoricalAwards=false
• 关键词: RUI; Sortase; Mediated; Ligations; Probing

The functions of most cellular proteins are understood to depend on their shape or structure. Many proteins, however, include parts where the structure is not well defined. The shape and thus the functions of these intrinsically disordered regions (IDRs) of proteins are difficult study. With this award, the Chemistry of Life Processes Program in the Chemistry Division is supporting the research of Dr. John Antos and Dr. Sergey Smirnov from Western Washington University to develop experimental methods to study the structures of IDRs. The research team is using use a splicing enzyme (sortase) to insert specific chemical tags into proteins that allow their shapes and changes to their shapes to be monitored by Nuclear Magnetic Resonance (NMR) spectroscopy. This project applies these methods to determine the key functional properties of dematin, a protein with a large IRD that helps control red blood cell shape and function. Results from these studies set the stage to study many types of IDRs in proteins that are prevalent across multiple kingdoms of life. The project prepares the next generation of scientists by training participating undergraduate and Masters level graduate students in state-of-the-art methods for analyzing protein structure and function. The project provides research opportunities to engage and attract underrepresented groups and high school students into the natural sciences. In addition, Drs. Antos and Smirnov incorporate authentic research experiences into Course-based Undergraduate Research Experiences (CUREs) for biochemistry courses. Approximately one-third of eukaryotic proteins contain within their polypeptide sequences sizable intrinsically disordered regions (IDRs), which are critical for a variety of cellular functions. Many of these IDRs are large (hundreds of residues), which makes the site-specific study of these dynamic protein segments and their binding interfaces technically challenging. For example, the application of NMR, one of the preferred methods for studying IDRs, is hampered by severe spectral overlap of resonances originating from large IDRs. Similarly, the targeted insertion of functional labels such as spectroscopic reporters or post-translational modifications is often impossible due to competing labeling of multiple identical sites in extended IDR sequences. This project addresses such problems via robust and efficient site-specific sortase-mediated ligation strategies tuned for large IDR-containing substrates. This includes the development of guidelines for the use of site-specific ligation strategies in the context of large IDR-containing protein substrates. The set of methods developed lead to the generation of derivatives of the erythrocyte-controlling protein dematin segmentally labeled with NMR-active isotopes (15N and 13C). Such derivatives enable characterization of a crucial regulatory intramolecular binding interface involving the large, 315-residue IDR that is central for dematin function and regulation.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.

大多数细胞蛋白质的功能取决于它们的形状或结构。然而，许多蛋白质包含结构不明确的部分。蛋白质的这些内在无序区（IDRs）的形状和功能是很难研究的。有了这个奖项，化学部的生命过程化学项目正在支持西华盛顿大学的约翰·安托斯博士和谢尔盖·斯米尔诺夫博士的研究，以开发研究idr结构的实验方法。研究小组正在使用剪接酶（分选酶）将特定的化学标签插入蛋白质中，使其形状和形状的变化能够通过核磁共振（NMR）光谱学进行监测。该项目应用这些方法来确定失蛋白的关键功能特性，失蛋白是一种具有大IRD的蛋白质，有助于控制红细胞的形状和功能。这些研究的结果为研究在多个生命王国中普遍存在的蛋白质中的许多类型的idr奠定了基础。该项目通过培训参与的本科生和硕士研究生，用最先进的方法分析蛋白质结构和功能，为下一代科学家做好准备。该项目提供研究机会，吸引代表性不足的群体和高中生参与自然科学。此外，博士。安托斯和斯米尔诺夫将真实的研究经历纳入生物化学课程的基于课程的本科生研究经历（CUREs）。大约三分之一的真核蛋白在其多肽序列中含有相当大的内在无序区（IDRs），这对各种细胞功能至关重要。这些idr中的许多都很大（数百个残基），这使得这些动态蛋白质片段及其结合界面的位点特异性研究在技术上具有挑战性。例如，研究idr的首选方法之一核磁共振的应用受到大型idr产生的严重的频谱重叠的阻碍。同样，由于扩展IDR序列中多个相同位点的竞争性标记，诸如光谱报告或翻译后修饰等功能标签的靶向插入通常是不可能的。该项目通过针对大型含idr的底物调整的强大而有效的位点特异性排序酶介导的连接策略来解决这些问题。这包括在含有大量idr的蛋白质底物的情况下，制定使用位点特异性连接策略的指南。开发的一套方法导致生成红细胞控制蛋白的衍生物，用核磁共振活性同位素（15N和13C）分段标记。这些衍生物能够表征一个关键的调节分子内结合界面，涉及大的315个残基IDR，它是失活蛋白功能和调节的核心。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。