RUI: Intein Structure and Catalysis of Protein Splicing and Hedgehog Autoprocessing: An Integrated Research and Education Program

智力价值这个项目将研究蛋白质剪接的化学机制。蛋白质剪接是一种介入性多肽或内含子催化自身从侧翼多肽或外显子上移除的方法。内含子还连接外显子片段。该研究项目将研究以下内容：1.极端嗜热者的内含素的活性如何受到其异常结构稳定性的影响。研究方法将考察内含素如何平衡高温下稳定的需要和足够灵活以具有催化活性的需要；2.内含子中的保守残基催化反应的每一步的方式，从而深入了解酶如何在没有辅助因子的情况下催化和协调单个活性部位的多步骤反应；3.氨基酸天冬酰胺或谷氨酰胺的环化催化，与多肽键断裂相连。这在蛋白质化学中是一种不寻常的反应，将在内含子的背景下以及通过模型肽实验和计算模拟的组合来研究；4.内含子活性的调节，无论是温度还是氧化状态。这一调控过程可能会提示内含子如何控制其侧翼、中断的蛋白质的活性；5.在结构和序列上与内含子相似的刺猬结构域的自动处理，包括保守的残基和胆固醇的结合可能如何影响反应的初始步骤。蛋白质剪接的这些方面将在适合本科生的项目中结合使用结构、生物物理、生化和计算技术进行研究。广泛影响该研究项目将对本科生的职业生涯产生重大影响。本科生将进行实验，在国家会议和同行评议的出版物上展示结果，并通过密切的指导，为成为STEM未来的领导者做准备。圣十字学院生物化学专业的学生将被招募来参与这个项目。除了研究活动外，学生们还将通过举办面向广大受众的讲座和参与科学大使学生推广计划，在社区中发起推广活动。该项目将通过伍斯特管道招募当地学生进入实验室，该项目是与马萨诸塞大学医学院合作开展的一个项目

Intellectual MeritThis project will study the chemical mechanism of protein splicing. Protein splicing is the means by which an intervening polypeptide, or intein, catalyzes its own removal from flanking polypeptides, or exteins. The intein also links the extein segments. The research project will investigate the following: 1. How the activity of an intein from an extreme thermophile is influenced by its unusual structural stability. The investigative approaches will examine how the intein balances the need to be stable at high temperatures with the need to be sufficiently flexible to be catalytically active; 2. The manner in which conserved residues in the intein catalyze each step of the reaction, providing insight into how enzymes catalyze and coordinate multi-step reactions at a single active site without cofactors; 3. The catalysis of cyclization of the amino acids asparagine or glutamine, coupled to peptide bond cleavage. This is an unusual reaction in protein chemistry which will be studied in the context of an intein as well as with a combination of model peptide experiments and computational modeling; 4. The regulation of intein activity, either by temperature or oxidation state. This process of regulation might suggest how inteins could control the activity of their flanking, interrupted proteins; 5. The autoprocessing of hedgehog-like domains, which resemble inteins in structure and sequence, including how conserved residues and the binding of cholesterol may influence the initial step of the reaction. These aspects of protein splicing will be investigated with the use of a combination of structural, biophysical, biochemical and computational techniques in projects suitable for undergraduates.Broader ImpactThe research program will have a significant impact on the careers of undergraduate research students. The undergraduates will perform the experiments, present the results at national meetings and in peer-reviewed publications, and, through close mentoring, prepare to be future leaders in STEM. Students from the Biochemistry Concentration at the College of Holy Cross will be recruited to work on this project. In addition to research activities, the students will sponsor outreach in the community by hosting lectures aimed at broad audiences and by participating in the Science Ambassadors student outreach program. The project will broaden access to STEM by recruiting local students to the lab through the Worcester Pipeline which is a program that is conducted in conjunction with the University of Massachusetts Medical School