Protein Unfolding and Refolding by Multidimensional Spectroscopy

Abstract

Unfolding and refolding studies using chemical denaturants such as urea or guanidine hydrochloride have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. The simplest and most frequently used approach to analyse denaturant-induced unfolding is based on the assumption that the free energy of unfolding in the absence of denaturant can be extrapolated from free energy values measured in the presence of finite denaturant concentrations. However, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation procedure, which results in poor reliability of the best-fit parameter values.Here we show that this limitation can be overcome by combining multiple spectroscopic signals - including fluorescence, circular dichroism, and absorbance - recorded at many different wavelengths. Modern spectrometers can collect all of these signals in a quasi-simultaneous and fully automated way. We have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. Compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the reliability of the free energy value by an order of magnitude (in terms of the width of the confidence intervals). We exemplify and validate this novel approach using some representative proteins and explain how it can be exploited to quantify subtle changes in protein stability which have thus far remained elusive.