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

本项目将研究蛋白质剪接的化学机制。蛋白质剪接是一种手段，通过干预多肽，或内部蛋白，催化其自身从侧面多肽，或外链蛋白去除。内链也连接外链段。该研究项目将调查以下内容：极端嗜热菌的蛋白活性如何受到其不寻常的结构稳定性的影响。研究方法将研究肠道如何在高温下保持稳定的需要与足够灵活的催化活性的需要之间取得平衡；2. 蛋白中的保守残基催化反应的每一步的方式，提供了酶如何在没有辅助因子的情况下在单个活性位点催化和协调多步反应的见解；3. 天冬酰胺或谷氨酰胺的环化催化，偶联到肽键的裂解。这是蛋白质化学中的一种不寻常的反应，将在蛋白质的背景下进行研究，并结合模型肽实验和计算建模；4. 通过温度或氧化状态对蛋白质活性的调节这一调节过程可能表明，蛋白是如何控制其侧翼被打断的蛋白的活性的；5. 在结构和序列上类似于蛋白的刺猬结构域的自动处理，包括保守残基和胆固醇的结合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。更广泛的影响研究计划将对本科研究生的职业生涯产生重大影响。本科生将进行实验，在全国会议和同行评审的出版物上展示结果，并通过密切的指导，为成为STEM领域未来的领导者做好准备。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听众的讲座和参加科学大使学生外展计划来赞助社区外展活动。该项目将通过伍斯特管道（Worcester Pipeline）招募当地学生到实验室，从而扩大STEM的使用范围。伍斯特管道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