Resolving multiple protein conformers in equilibrium unfolding reactions: a time-resolved emission spectroscopic (TRES) study of Azurin.

Guptasarma, Purnananda (1997) Resolving multiple protein conformers in equilibrium unfolding reactions: a time-resolved emission spectroscopic (TRES) study of Azurin. Biophysical chemistry, 65 (2-3). pp. 221-8. ISSN 0301-4622

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Abstract

Unlike steady-state spectrofluorimetry, time-resolved emission spectroscopy (TRES) can resolve emissions from fluorophores with similar quantum yields and overlapping steady-state emission spectra. Time-resolved emission studies of the protein-intrinsic fluorophore, tryptophan (Trp), can thus potentially be used to examine protein conformational heterogeneity in solution, as well as to investigate the existence of populated intermediate structural states in equilibrium unfolding reactions of single-tryptophan proteins. Here, the single-Trp copper protein, azurin, is examined in various concentrations of guanidine hydrochloride (GdnCl) with its disulphide bond in an intact state. Interestingly, multiple envelopes of Trp emission are observed in all TRES spectra acquired, instead of just two emission envelopes (corresponding to the native and unfolded states) expected from two-state unfolding. These envelopes appear to be centred around the same set of emission wavelengths in different TRES spectra, and only intensities and decay rates vary with the concentration of denaturant used. This suggests that structural states representing different levels of exposure of Trp to the aqueous solvent might, in fact, be populated at equilibrium during the unfolding of azurin by GdnCl.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Science.
Uncontrolled Keywords:	Fluorescence decay time-resolved emission; Protein conformation; Protein folding; Structural intermediates
Subjects:	Q Science > QD Chemistry
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	27 Jan 2012 14:57
Last Modified:	02 Apr 2012 09:22
URI:	http://crdd.osdd.net/open/id/eprint/770

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