RUI: Protein Splicing and the Structure and Stability of Thermophilic Inteins

该研究计划的目标是研究嗜热生物的蛋白质化学。嗜热微生物是生活在非常热的地方的微生物，通常处于非常高的压力下，例如深海热喷口。该计划将研究这些蛋白质如何在这种极端条件下发挥作用，这可能会展开或阻止在更适宜的条件下生长的生物体中蛋白质的活性。这将使我们能够深入了解蛋白质在保持化学活性的同时稳定其结构的策略。特别是，该计划将研究一类称为内含肽的酶（促进化学反应的蛋白质催化剂）。内含肽干扰其他蛋白质，必须促进它们自身的去除，以便被干扰的蛋白质能够发挥作用。了解这种反应是如何调节的，可能会提示剪接反应的作用，以及控制基因表达的潜在新方法。内含肽和更广泛的嗜热酶都具有有用的生物技术应用，对结构和功能的更深入理解将改善这些工具。该计划的更广泛的影响包括下一代科学家和STEM领导者的发展，通过直接培训本科研究生，通过推广到伍斯特社区最年轻的科学家，并通过增加科学研究的机会，代表团体都在伍斯特学生社区和圣十字一年级的学生。研究计划旨在了解的机制，蛋白质剪接，由内含肽催化的翻译后修饰。它将使用X射线晶体学和核磁共振结构和动力学实验，结合生物化学技术，以原子级分辨率研究有关催化机制的未解决问题。它将使用一个生物物理模型肽系统，结合计算技术，研究侧链环化耦合到肽键切割。它将研究嗜热内含肽的结构如何影响它们的活性和构象灵活性，并研究剪接的生理作用。此外，该计划将对我们理解酶如何在单个活性位点催化多步反应以及嗜热酶如何通过蛋白质动力学平衡活性和稳定性产生重大影响。

The goal of the research program is to study the chemistry of proteins from thermophilic organisms. Thermophiles are microorganisms that live in places that are very hot and are often under very high pressure, such as deep-sea thermal vents. The program will investigate how these proteins can function at such extreme conditions, which could unfold or prevent the activity of proteins from organisms that grow under more hospitable conditions. This would allow insight into strategies that proteins use to stabilize their structure while remaining chemically active. In particular, the program will examine a class of enzymes (protein catalysts that facilitate chemical reactions) called inteins. Inteins interrupt other proteins, and must promote their own removal so that the interrupted proteins can function. Learning how this reaction is regulated may suggest roles for the splicing reaction and potentially new ways in which gene expression can be controlled. Inteins, and thermophilic enzymes more broadly, both have useful biotechnology applications, and a deeper understanding of structure and function will improve these tools. The broader impact of the program includes the development of the next generation of scientists and leaders in STEM, through direct training of undergraduate research students, through outreach to the youngest scientists in the Worcester community, and through increasing access to scientific research for under-represented groups both in the Worcester student community and with Holy Cross first year students.The research program seeks to understand the mechanism of protein splicing, a post-translational modification catalyzed by an intein. It will study unresolved questions about the catalytic mechanism at atomic-level resolution using both X-ray crystallography and NMR structure and dynamics experiments coupled with biochemical techniques. It will use a biophysical model peptide system, integrated with computational techniques, to investigate side chain cyclization coupled to peptide bond cleavage. It will examine how the structure of thermophilic inteins influences their activity and conformational flexibility, and investigate the physiological role of splicing. In addition, the program will have significant impact on our understanding of how enzymes catalyze multi-step reactions at a single active site and how thermophilic enzymes balance activity and stability through protein dynamics.