Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength

Abstract

The fluorescence of tryptophan is used as a signal to monitor the unfolding of proteins, in particular the intensity of fluorescence and the wavelength of its maximum λmax. The law of the signal is linear with respect to the concentrations of the reactants for the intensity but not for λmax. Consequently, the stability of a protein and its variation upon mutation cannot be deduced directly from measurements made with λmax. Here, we established a rigorous law of the signal for λmax. We then compared the stability ΔG(H2O) and coefficient of cooperativity m for a two-state equilibrium of unfolding, monitored with λmax, when the rigorous and empirical linear laws of the signal are applied. The corrective terms involve the curvature of the emission spectra at their λmax and can be determined experimentally. The rigorous and empirical values of the cooperativity coefficient m are equal within the experimental error for this parameter. In contrast, the rigorous and empirical values of the stability ΔG(H 2O) generally differ. However, they are equal within the experimental error if the curvatures of the spectra for the native and unfolded states are identical. We validated this analysis experimentally using domain 3 of the envelope glycoprotein of the dengue virus and the single-chain variable fragment (scFv) of antibody mAbD1.3, directed against lysozyme. © The Author 2005. Published by Oxford University Press. All rights reserved.