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

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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.