A method for determining states in the course of protein unfolding

Abstract

A simple model is presented for analyzing a set of spectra obtained from spectrophotometric study of protein titration. With this model one can determine the states of (probable) intermediates in the course of protein unfolding. The model is developed based on abundance of native state, intermediate(s) and denatured state, and their contributions to differential absorbance at selected wavelengths. The model is tested for the two-state unfolding of ribonuclease A by urea in formate buffer, and also for the three-state unfolding of a-lactalbumin by guanidine hydrochloride (GdnHCl) in phosphate buffer. It was demonstrated that unfolding of ribonuclease A is matched acceptably with the two-state model, while a-lactalbumin unfolding starts with a two-state mechanism (when [GdnHCl]>=1.6M) followed by a three-state pathway.