Collaborative Research: Engineering Hyperstable Enzymes via Computationally Guided Protein Stapling

酶是促进反应的蛋白质。在适度的温度下或响应于pH变化，酶将失去其三维结构，这导致其作为催化剂的活性损失。如果酶更稳定，那么它们可以在工业应用中得到更广泛的应用。这项研究项目将研究一种新的方法来设计蛋白质，其中化学连接将在酶的某些部分之间进行，也称为“分子钉合”。“这种方法可能会增加蛋白质在高温或化学物质存在下的稳定性。具有“斯台普斯”的新酶的活性应与原始酶相同。该项目的结果可能导致酶可以被设计用于制造药物或以更可持续的方式生产化学品。 该研究项目作为本科生和研究生进行尖端研究的培训基地，培养出一支熟练的STEM劳动力。研究人员将接待当地高中生，让他们体验酶和蛋白质的研究经验。 他们还将参与到社区大学的推广活动中。这个研究项目背后的想法是，酶可以通过基因编码的、氧化还原稳定的共价交联来稳定。一种新的计算设计方法，通过共价钉合蛋白质稳定将开发通过迭代设计-构建-测试周期，使用卡宾转移酶作为测试床。这种方法的范围将扩展到不同的钉合策略，以最大限度地提高目标酶的热力学和动力学稳定性，从而导致超稳定。计算方法的开发将通过功能测定和互补生物物理技术对钉合酶变体进行详细研究来指导。这种方法的一般性将通过其应用于具有生物技术重要性的不同酶的快速有效的热稳定来证明。据设想，这种方法将广泛适用于酶和蛋白质，提供了一个有效的，并可能是一般性的蛋白质hyperstabilization.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

Enzymes are proteins that facilitate reactions. At modest temperatures or in response to pH changes, enzymes will lose their three-dimensional structure which leads to a loss in their activity as catalysts. If enzymes were more stable, then they could be used more widely in industrial applications. This research project will investigate a new method for designing proteins in which chemical links will be made between certain parts of the enzyme, also called "molecular stapling." This method might increase the stability of proteins at elevated temperatures or in the presence of chemicals. The activity of the new enzyme with "staples" should be unchanged from the original enzyme. The results of this project could lead to enzymes that could be designed to manufacture pharmaceuticals or to produce chemicals in a more sustainable manner. The research project serves as a training ground for undergraduate and graduate students to perform cutting edge research, leading to a skilled STEM workforce. The researchers will host local high school students for a research experience on enzymes and proteins. They will also participate in outreach to area community colleges.The idea behind this research project is that enzymes can be stabilized by genetically encoded, redox-stable covalent crosslinks. A novel computational design method for protein stabilization via covalent stapling will be developed via iterative design-build-test cycles, using a carbene transferase as testbed. The scope of this methodology will be extended to different stapling strategies for maximizing both thermodynamic and kinetic stability of the target enzyme, leading to hyperstabilization. Computational methodology development will be guided by detailed investigations of the stapled enzyme variants via functional assays and complementary biophysical techniques. The generality of this approach will be demonstrated through its application for the rapid and efficient thermostabilization of different enzymes of biotechnological importance. It is envisioned that this method will be broadly applicable to enzymes and proteins, providing an effective, and potentially general strategy for protein hyperstabilization.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.