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PEDS Advance Access published online on August 8, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi046
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received June 27, 2005
Accepted July 1, 2005

Article

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

Elodie Monsellier 1 and Hugues Bedouelle 1*

1 Unit of Molecular Prevention and Therapy of Human Diseases (CNRS FRE 2849), Institut Pasteur, 28 rue Docteur Roux, 75724 Paris Cedex 15, France

* To whom correspondence should be addressed.
Hugues Bedouelle, E-mail: hbedouel{at}pasteur.fr


  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 {lambda}max. The law of the signal is linear with respect to the concentrations of the reactants for the intensity but not for {lambda}max. Consequently, the stability of a protein and its variation upon mutation cannot be deduced directly from measurements made with {lambda}max. Here, we established a rigorous law of the signal for {lambda}max. We then compared the stability {Delta}G(H2O) and coefficient of cooperativity m for a two-state equilibrium of unfolding, monitored with {lambda}max, when the rigorous and empirical linear laws of the signal are applied. The corrective terms involve the curvature of the emission spectra at their {lambda}max 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 {Delta}G(H2O) 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.

Keywords: cooperativity; denaturant; dengue virus; envelope glycoprotein; free energy; scFv antibody fragment; unfolding.
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