Yeast Frataxin Is Stabilized by Low Salt Concentrations: Cold Denaturation Disentangles Ionic Strength Effects from Specific Interactions

Abstract

Frataxins are a family of metal binding proteins associated with the human Friedreich's ataxia disease. Here, we have addressed the effect of non-specifically binding salts on the stability of the yeast ortholog Yfh1. This protein is a sensitive model since its stability is strongly dependent on the environment, in particular on ionic strength. Yfh1 also offers the unique advantage that its cold denaturation can be observed above the freezing point of water, thus allowing the facile construction of the whole protein stability curve and hence the measurement of accurate thermodynamic parameters for unfolding. We systematically measured the effect of several cations and, as a control, of different anions. We show that, while strongly susceptible to ionic strength, as it would be in the cellular environment, Yfh1 stability is sensitive not only to divalent cations, which bind specifically, but also to monovalent cations. We pinpoint the structural bases of the stability and hypothesize that the destabilization induced by an unusual cluster of negatively charged residues favours the entrance of water molecules into the hydrophobic core, consistent with the generally accepted mechanism of cold denaturation.

Selective observation of the disordered import signal of a globular protein by in‐cell NMR: The example of frataxins

“…(A)]. Using the assignment recently obtained, the first 10 residues have low chemical shift indexes although not completely null. The chemical shifts of the corresponding in‐cell resonances are indistinguishable from these values indicating that the residues must have the same secondary structure in cell and in the lysate (Fig.…”

“…The two frataxin orthologues, cloned in pET21a vectors, were transformed in BL21(DE3) E. coli cells and selected for transformation in ampicillin plates, according to previously published protocols. 16,24 CyaY comprised the full-length 106 amino acid protein sequence, whereas the Yfh1 construct contained residues 522174 which corresponds to the mature form of Yfh1. 19 Val52 was mutated to a methionine for molecular biology purposes.…”

The non-uniform early structural response of globular proteins to cold denaturing conditions: A case study with Yfh1

“…This observation corroborates previous results. 14,39 In order to understand the local hydration effects upon loss of structure, we have calculated the IR absorption spectra for representative beta (S2, S4, and S5) and helical (H1) domains. A detailed inspection using IR spectra of these individual beta-sheet domains show similar trends of spectral red shifts as observed for the full protein, whereas a red shift is not observed in the calculated IR spectra of the representative helical domain of the protein upon lowering the temperature.…”

“…24,30,34,[37][38][39][40] It was found that at the temperatures at which heat and cold denaturation are initiated (304 and 280 K, respectively), 34 the two denatured states exhibit similar conformational characteristics. 38 However, residual structural characteristics, compactness, and the extent of hydration of these states diverge as the temperatures of complete unfolding are approached.…”

“…Very recent results have indicated that influx of solvent molecules into the hydrophobic core of Yfh1 arising due to electrostatic repulsion between the similarly charged helices could be a plausible reason for its cold denaturation. 39 In this study, using molecular dynamics (MD) simulations, we have attempted to correlate the nature of protein surface hydration with the earliest structural response of Yfh1 to cold denaturing conditions. While fully atomistic, submicrosecond simulations are insufficient to unravel the complete unfolding pathway, they demonstrate the non-uniform structural response of the different domains of Yfh1 to the thermal conditions.…”