Phi-Analysis of CusF: Alanine-scanning Mutagenesis to Reveal the Folding Transition State

Do proteins follow the same folding mechanism and are they stabilized when introduced to structural mutations? How does the transition state change when introduced to these mutations? The transition state structure tells us whether a protein is folded or unfolded as the reaction proceeds. CusF, a small, periplasmic Ag(I) and Cu(I) chaperone protein found in E. Coli is used as a model to address this. Phi-analysis is used as a means of determining the transition state of CusF wild-type protein and the mutated residues’ contribution to the folding transition state from the free energy of the unfolded state. Assumptions that can be made from φ-analysis are that the folding pathway will not be significantly altered but the folding energies will be. A φ-value of 1 implies that the degree to which the transition state is stabilized is equal to the folded state stabilization and the structure in the transition state resembles the native state. Through alanine-scanning mutagenesis, a simple technique in the determination of the functional role of protein residues, reaction rates were measured. We expect alanine mutagenesis in the beta-barrel to stabilize folding and increase its free energy, resulting in a slower rate of folding. About 76% of CusF’s amino acids have been mutated to alanine in order to stabilize it and the subset chosen for mutations with greatest influence on folding occurs within the OB-barrel to stabilize rate of unfolding. Future work includes complete alanine-scanning to the protein and determining the complete reaction mechanism for CusF.