6] Protein engineering in analysis of protein folding pathways and stability

Abstract

Publisher Summary This chapter discusses the way the folding process of a protein can be examined using a combination of equilibrium and kinetic experiments and protein engineering. A specific interaction that stabilizes the folded structure is deleted by a small nondisruptive mutation. The energetic contribution of the interaction to the stability of folding of the protein is measured from equilibrium unfolding experiments, usually by urea-mediated denaturation. The next step is to make kinetic measurements on the folding and unfolding of wild type and mutant proteins. This can be used to measure the effects the mutations have on the stability of other forms of the protein, such as the transition state of the rate-determining step in unfolding and, possibly, intermediates present on the folding pathway. This is performed by constructing the free energy profile for the reaction. The energy of the transition state is determined from the unfolding rate constant using transition state theory and the energy of intermediates by the ratio of unfolding and refolding constants. In practice, transition state theory is used quantitatively only to measure the differences in energy between transition states of wild-type and mutant enzymes and not the absolute value.