Collaborative Research : Engineering Hyperstable Enzymes via Computationally Guided Protein Stapling

酶是促进反应的蛋白质。在适度的温度或对pH变化的反应中，酶将失去其三维结构，从而导致其作为催化剂的活性丧失。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。这项研究项目将探索一种设计蛋白质的新方法，在这种方法中，酶的某些部分之间将建立化学联系，也被称为“分子装订”。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。带有“订书钉”的新酶的活性应与原酶保持不变。这个项目的结果可能会导致酶的设计，可以用来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训练场，培养了一支熟练的STEM劳动力队伍。研究人员将招待当地高中生体验酶和蛋白质的研究。他们还将参与到地区社区大学的外联活动。这项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键来稳定。以卡宾转移酶为试验床，通过迭代设计-构建-测试循环，开发了一种新的通过共价装订来稳定蛋白质的计算设计方法。这一方法的范围将扩展到不同的装订策略，以最大化目标酶的热力学和动力学稳定性，从而导致超稳定。计算方法的发展将通过功能分析和补充生物物理技术对装订酶变异体的详细调查来指导。这种方法的通用性将通过其在快速和有效地稳定具有生物技术重要性的不同酶上的应用来展示。据设想，这种方法将广泛适用于酶和蛋白质，为蛋白质超稳定提供一个有效的、潜在的一般策略。该奖项反映了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。

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.