Modulation of Protein Folding Energy Landscape through Chain Connectivity and Ligand Binding
Abstract
Protein is the building block of all living organisms. To achieve the appropriate function, a protein needs to effectively fold to its three-dimensional conformation. Protein misfolding can cause severe disease conditions such as neurodegenerative diseases and cystic fibrosis. The amino acid sequence and the surrounding environment of a protein determine the folding process. This dissertation elucidates the effect of chain connectivity and ligand binding on protein folding. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated the effect of circular permutation on partial unfolding. Our finding revealed that the location of the termini relative to the unfolded region of the partially unfolded form of a protein determines the energetics of partial unfolding. In this dissertation, the mechanism of cofactor-mediated folding of E. coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also investigated. An initial investigation of the effect of the structural fragments of NAD+ on the folding of GAPDH revealed that cofactor NAD+ facilitates the folding of GAPDH through its adenosine moiety. The adenosine moiety of NAD+ selectively stabilizes the transition state of a rate-limiting step, which has a partially folded structure. A mutagenesis study of the binding pocket was employed to map the residues responsible for the effect of NAD +. The mutagenesis study elucidated that the adenine-binding subsite of the binding pocket is the determinant for the effect of NAD+ on GAPDH folding. Furthermore, the adenine-binding subsite and pyrophosphate-binding subsite of the binding pocket is folded in the partially unfolded transition state, but the nicotinamide-binding subsite is not. A novel mechanism of cofactor-mediated folding was proposed based on the structure-activity study of NAD+ and mutagenesis study of GAPDH.
Degree
Ph.D.
Advisors
Park, Purdue University.
Subject Area
Biophysics
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