Use of quencher to decode three-state nature of unfolding of proteins from fluorescence intensity–temperature profiles

Three-state unfolding profiles obtained using spectroscopic techniques can generally be fitted using two-state equation, making it difficult to make a distinction between two-state and three-state transition. Simulations of fluorescence intensity–temperature profile for a three-state transition and subsequent fitting using two-state equation suggest that it is easier to decipher three-state nature of protein unfolding from temperature induced unfolding profile rather than denaturant induced unfolding profile. However, there are some extreme cases where it is difficult to predict the three-state transition even from temperature induced protein unfolding profiles. The simulation and the analysis of unfolding data of bovine serum albumin in the absence and presence of sodium dodecyl sulfate suggest that the addition of a fluorescence quencher helps in differentiating a two-state model with three-state model in such extreme cases. In addition, it helps to distinguish two different three-state models, one with higher first enthalpy ΔHm1 and other with lower ΔHm1.