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Trapping a salt-dependent unfolding intermediate of the marginally stable protein Yfh1.

ABSTRACT: Yfh1, the yeast ortholog of frataxin, is a protein of limited thermodynamic stability which undergoes cold denaturation at temperatures above the water freezing point. We have previously demonstrated that its stability is strongly dependent on ionic strength and that monovalent or divalent cations are able to considerably stabilize the fold. Here, we present a study of the folded state and of the structural determinants that lead to the strong salt dependence. We demonstrate by nuclear magnetic resonance that, at room temperature, Yfh1 exists as an equilibrium mixture of a folded species and a folding intermediate in slow exchange equilibrium. The equilibrium completely shifts in favor of the folded species by the addition of even small concentrations of salt. We demonstrate that Yfh1 is destabilized by a localized energetic frustration arising from an "electrostatic hinge" made of negatively charged residues mapped in the ?-sheet. Salt interactions at this site have a "frustration-relieving" effect. We discuss the consequences of our findings for the function of Yfh1 and for our understanding of protein folding stability.

SUBMITTER: Vilanova B

PROVIDER: S-EPMC4428383 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Trapping a salt-dependent unfolding intermediate of the marginally stable protein Yfh1.

Vilanova Bartolomé B Sanfelice Domenico D Martorell Gabriel G Temussi Piero A PA Pastore Annalisa A

Frontiers in molecular biosciences 20140930

Yfh1, the yeast ortholog of frataxin, is a protein of limited thermodynamic stability which undergoes cold denaturation at temperatures above the water freezing point. We have previously demonstrated that its stability is strongly dependent on ionic strength and that monovalent or divalent cations are able to considerably stabilize the fold. Here, we present a study of the folded state and of the structural determinants that lead to the strong salt dependence. We demonstrate by nuclear magnetic ...[more]

PMID: 25988154

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Project description:Reversible unfolding of ovomucoid by guanidine hydrochloride, as followed by viscosity and difference-spectral measurements at 25 degrees C, pH6, occurred in two distinct steps involving at least three major conformational states, namely the native, intermediate and completely denatured states, occurring respectively in 60mm-sodium phosphate buffer, 3.5m-guanidine hydrochloride and 6m-guanidine hydrochloride. The overall native conformation of ovomucoid, as indicated by its intrinsic viscosity (5.24ml/g) and gel-filtration behaviour, differs significantly from that of a typical globular protein. Exposures of tyrosine residues in native ovomucoid measured by difference spectroscopy following perturbation with glycerol, ethylene glycol and dimethyl sulphoxide were, respectively, 0.42, 0.56 and 0.57. Of the exposed phenolic groups only one titrated normally (pK(int.), 9.91, electrostatic-interaction factor, w, 0.04). Results on difference spectra, solvent perturbation, phenolic titration and intrinsic viscosity (7.4ml/g) taken together showed that, although ovomucoid in 3.5m-guanidine hydrochloride was significantly unfolded, it retained a degree of native structure, removable with 6m-guanidine hydrochloride. In the latter, all the six tyrosine residues were available for titration, and the intrinsic viscosity of ovomucoid increased to 9.4ml/g. Furthermore, the characteristic fine structures in circular-dichrosim spectra of ovomucoid, associated with the elements of native structure, were abolished in 6m-guanidine hydrochloride, suggesting that the completely denatured state is structureless and presumably behaves as a cross-linked random coil. The latter state has been shown by analysis of the results on guanidine hydrochloride-dependence of the transition, intermediateright harpoon over left harpoondenatured, to be less stable than the intermediate state under native conditions by about 46kJ/mol at 25 degrees C. Attempts have been made to interpret the above results in the light of available information on the amino acid sequence of ovomucoid.

Dataset Information

Trapping a salt-dependent unfolding intermediate of the marginally stable protein Yfh1.

Publications

Trapping a salt-dependent unfolding intermediate of the marginally stable protein Yfh1.

Similar Datasets

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