PECASE: The Significance of Intermediate Structures in Protein Folding and Assembly

Colon MCB 9984913本研究的目的是使用二聚体蛋白质Fis（倒置刺激因子）作为模型系统来研究蛋白质折叠和寡聚化中动力学中间体的意义。两个主要目的是（1）调节Fis的折叠途径以控制动力学中间体的群体，和（2）确定中间体的存在或不存在如何影响Fis的稳定性及其折叠动力学。将研究特定残基变化对对应于三个Fis亚结构域的肽的结构和稳定性的影响。然后，这些变化将被整合到全长蛋白质中，一次一个结构域或组合以改变折叠中间体的存在。将使用荧光和圆二色性（CD）光谱结合快速混合（停流）方法分别监测Fis和Fis肽的二级和三级结构。尽管进行了大量研究，但蛋白质的氨基酸序列决定其3D结构的机制仍然知之甚少，而3D结构对功能至关重要。破译包含在氨基酸序列中的“折叠密码”可以在生物技术中产生许多重要的应用，包括预测蛋白质的三维结构和合理设计具有所需折叠和功能的蛋白质。该项目将使用光学光谱来研究蛋白质折叠研究中经常观察到的蛋白质中间构象状态是否是正确和有效折叠所必需的。该项目的教育部分涉及对本科生和高中生的培训和辅导，重点是代表性不足的少数群体。预计通过研究机会，这些学生将获得培训，经验和知识，以考虑科学作为职业选择。

Colon MCB 9984913The goal of this research is to use the dimeric protein Fis (factor for inversion stimulation) as a model system to investigate the significance of kinetic intermediates in protein folding and oligomerization. Two main objectives are (1) to modulate the folding pathway of Fis to control the population of kinetic intermediate(s), and (2) to determine how the presence or absence of intermediates affects the stability of Fis and its folding kinetics. The effect of specific residue changes on the structure and stability of peptides corresponding to the three Fis sub-domains will be studied. These changes will then be incorporated into the full-length protein, one domain at a time or in combination to alter the presence of folding intermediates. Fluorescence and circular dichroism (CD) spectroscopies in conjunction with rapid mixing (stopped-flow) methods will be used to monitor the secondary and tertiary structure, respectively, of Fis and Fis peptides. Despite much research, the mechanism by which the amino acid sequence of a protein determines its 3D structure, which is essential for function, is still poorly understood. Deciphering the "folding code" contained in the amino acid sequence could result in many significant applications in biotechnology, including the prediction of a protein's three dimensional structure and the rational design of proteins with a desired fold and function. This project will use optical spectroscopy to investigate whether protein intermediate conformational states that are often observed in protein folding studies are required for correct and efficient folding. The educational component of this project involves the training and mentoring of undergraduate and high school students with emphasis on underrepresented minorities. It is anticipated that through research opportunities, these students will obtain the training, experience, and knowledge to consider science as a career choice.