Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength.
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 DeltaG(H(2)O) 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 DeltaG(H(2)O) 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.
Monsellier
Elodie
E
Unit of Molecular Prevention and Therapy of Human Diseases (CNRS FRE 2849), Institut Pasteur, 28 rue Docteur Roux, 75724 Paris Cedex 15, France.
Bedouelle
Hugues
H
D003716:Dengue Virus; D005453:Fluorescence; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008722:Methods; D009113:Muramidase / Q000276:immunology; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014759:Viral Envelope Proteins / Q000737:chemistry
D007135:Immunoglobulin Variable Region; D014759:Viral Envelope Proteins; D014364:Tryptophan; D009113:Muramidase