Stability of proteins with multi-state unfolding behavior

Abstract

A new model used to calculate the free energy change of protein unfolding is presented. In this model, proteins are considered to be composed of structural elements. The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method. If a protein consists of the same structural elements, its unfolding will displays a two-state process, and only the average structural element free energy change 〈ΔG element 0 (H2O)〉 can be measured. If protein consists of completely different structural elements, its unfolding will show a multi-state behavior. When a protein consists of n structural elements its unfolding will shows a (n+1)-state behavior. A least-squares fitting can be used to analyze the contribution of each structural element to the protein and the free energy change of each structural element can be obtained by using linear extrapolation to zero denaturant concentration, not to the start of each transition. The measured ΔG protein 0 (H2O) is the sum of the free energy change for each structural element. Using this new model, we can not only analyze the stability of various proteins with similar structure and similar molecular weight, which undergo multi-state unfolding processes, but also compare the stability of proteins with different structures and molecular weights using the average structural element free energy change 〈ΔG element 0 (H2O)〉. Although this method cannot completely provide the exact free energy of proteins, it is better than current methods.