Are there equilibrium intermediate states in the urea-induced unfolding of hen egg-white lysozyme? † (1997).

Content Provider	CiteSeerX
Author	Ibarra-Molero, Beatriz Sanchez-Ruiz, Jose M.
Abstract	conditions have attracted much attention as plausible models for the kinetic intermediates transiently populated in the refolding kinetic pathways. Hen egg-white lysozyme is often considered as a typical example of close adherence to the equilibrium, two-state unfolding mechanism. However, recent smallangle X-ray scattering studies suggest that an equilibrium intermediate state is significantly populated in the urea-induced unfolding of this protein at moderately acidic pH. In this work, we analyze the ureainduced unfolding of hen egg-white lysozyme on the basis of steady-state fluorescence measurements, characterization of the folding-unfolding kinetics, double-jump unfolding assays for the amount of native protein, and double-jump refolding assays for the amount of unfolded protein. Our results do not provide support for the presence of an intermediate state and, in particular, disfavor that the following two types of intermediates be significantly populated at equilibrium: (1) intermediates showing a substantial quenching of the tryptophan fluorescence (such as that observed in the transient intermediates found in the refolding kinetic pathway under strongly native conditions) and (2) associating intermediates. Also, the deconvolution of the radius of gyration unfolding profile by using the values for the amount of native state derived from our double-jump unfolding assays is consistent with a two-state unfolding equilibrium and suggests, furthermore, that, in this case, large alterations in the average structure of the unfolded ensemble do not take place in response to changes in urea concentration. This work points up possible pitfalls in the experimental detection of equilibrium folding intermediates and suggests procedures to circumvent them.
File Format	PDF
Publisher Date	1997-01-01
Access Restriction	Open
Subject Keyword	Hen Egg-white Lysozyme Urea-induced Unfolding Equilibrium Intermediate State Double-jump Unfolding Assay Refolding Kinetic Pathway Equilibrium Folding Intermediate Acidic Ph Native Condition Intermediate State Substantial Quenching Native Protein Two-state Unfolding Equilibrium Urea Concentration Double-jump Refolding Assay Steady-state Fluorescence Measurement Unfolded Protein Average Structure Possible Pitfall Plausible Model Native State Typical Example Ureainduced Unfolding Tryptophan Fluorescence Unfolded Ensemble Two-state Unfolding Mechanism Folding-unfolding Kinetics Kinetic Intermediate Experimental Detection Close Adherence Recent Smallangle X-ray Much Attention Large Alteration
Content Type	Text