Unfolding of apomyoglobin from Aplysia limacina: the effect of salt and pH on the cooperativity of folding.

I<-->U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (delta Gw(app) and solvent accessibility (n(app)) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in delta G(w(app)) occur without changes in n(app). The change in free energy of folding of I<-->F (delta Gw(F/I)) decreases to 0 at pH 4 as expected from the acid unfolding curve. delta Gw(I/U) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n(app) although there is no change in the alpha-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour. -->
Substance Nomenclature: 0 (Apoproteins)
0 (Myoglobin)
0 (apomyoglobin)
660YQ98I10 (Potassium Chloride)
Entry Date(s): Date Created: 19980206 Date Completed: 19980210 Latest Revision: 20131121
Update Code: 20231215
DOI: 10.1006/jmbi.1997.1409
PMID: 9451445
Autor: Staniforth RA; Dipartimento di Scienze Biochimiche, A. Rossi Fanelli, Istituto Pasteur-Fondazione Cenci Bolognetti, Roma, Italy., Bigotti MG, Cutruzzolà F, Allocatelli CT, Brunori M
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 1998 Jan 09; Vol. 275 (1), pp. 133-48.
DOI: 10.1006/jmbi.1997.1409
Abstrakt: The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% alpha-helical under low salt conditions (2 mM citrate, 4 degrees C); the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F<-->I<-->U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (delta Gw(app) and solvent accessibility (n(app)) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in delta G(w(app)) occur without changes in n(app). The change in free energy of folding of I<-->F (delta Gw(F/I)) decreases to 0 at pH 4 as expected from the acid unfolding curve. delta Gw(I/U) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n(app) although there is no change in the alpha-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour.
Databáze: MEDLINE
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