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。蛋白质剪接是插入多肽或内含肽催化其自身从侧翼多肽或外显肽移除的手段。内含肽还连接外显肽片段。本研究计画将探讨以下几点：1.极端嗜热菌内含肽的活性如何受到其异常结构稳定性的影响。研究方法将检查内含肽如何平衡在高温下稳定的需要与足够灵活以具有催化活性的需要; 2.内含肽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因子的情况下在单个活性位点催化和协调多步反应的洞察; 3.催化天冬酰胺或谷氨酰胺的环化反应，并伴有肽键的断裂。这是蛋白质化学中的一种不寻常的反应，将在内含肽的背景下以及结合模型肽实验和计算建模进行研究; 4.内含肽活性的调节，通过温度或氧化状态。这种调节过程可能表明内含肽如何控制其侧翼中断蛋白的活性; 5.在结构和序列上类似内含肽的hedgehog样结构域的自动加工，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与结构，生物物理，生物化学和计算技术相结合，在适合本科生的项目中进行研究。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议的出版物上展示结果，并通过密切的指导，准备成为STEM的未来领导者。来自圣十字学院生物化学专业的学生将被招募参与这个项目。除了研究活动，学生将主办针对广大观众的讲座，并通过参加科学大使学生外展计划在社区推广赞助。该项目将通过与马萨诸塞州大学医学院联合开展的伍斯特管道项目，招募当地学生进入实验室，从而扩大STEM的使用范围

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