Using Intrinsic Fluorescence to Measure Protein Stability Upon Thermal and Chemical Denaturation

Understanding how point mutations affect the performance of protein stability has been the focus of several studies all over the years. Intrinsic fluorescence is commonly used to follow protein unfolding since during denaturation, progressive redshifts on tryptophan fluorescence emission are observed. Since the unfolding process (achieved by chemical or physical denaturants) can be considered as two-state N➔D, it is possible to utilize the midpoint unfolding curves (fU = 50%) as a parameter to evaluate if the mutation destabilizes wild-type protein. The idea is to determine the [D]_1/2 or T_m values from both wild type and mutant and calculate the difference between them. Positive values indicate the mutant is less stable than wild type.