Global analysis of three-state protein unfolding data.
A new method for analyzing three-state protein unfolding equilibria is described that overcomes the difficulties created by direct effects of denaturants on circular dichroism (CD) and fluorescence spectra of the intermediate state. The procedure begins with a singular value analysis of the data matrix to determine the number of contributing species and perturbations. This result is used to choose a fitting model and remove all spectra from the fitting equation. Because the fitting model is a product of a matrix function which is nonlinear in the thermodynamic parameters and a matrix that is linear in the parameters that specify component spectra, the problem is solved with a variable projection algorithm. Advantages of this procedure are perturbation spectra do not have to be estimated before fitting, arbitrary assumptions about magnitudes of parameters that describe the intermediate state are not required, and multiple experiments involving different spectroscopic techniques can be simultaneously analyzed. Two tests of this method were performed: First, simulated three-state data were analyzed, and the original and recovered thermodynamic parameters agreed within one standard error, whereas recovered and original component spectra agreed within 0.5%. Second, guanidine-induced unfolding titrations of the human retinoid-X-receptor ligand-binding domain were analyzed according to a three-state model. The standard unfolding free energy changes in the absence of guanidine and the guanidine concentrations at zero free-energy change for both transitions were determined from a joint analysis of fluorescence and CD spectra. Realistic spectra of the three protein states were also obtained.
Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331-7503, USA. harderm@onid.orst.edu
Mark E
ME
Harder
Max L
ML
Deinzer
Mark E
ME
Leid
Michael I
MI
Schimerlik
D000818:Animals; D002942:Circular Dichroism / Q000379:methods*; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D047490:Retinoid X Receptor alpha / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014508:Urea / Q000737:chemistry
D047490:Retinoid X Receptor alpha; D016173:Macrophage Colony-Stimulating Factor; D014508:Urea; D019791:Guanidine