Yeast frataxin is stabilized by low salt concentrations: cold denaturation disentangles ionic strength effects from specific interactions.

Domenico Sanfelice,Annalisa Pastore,Lorenzo Di Bari,Rita Puglisi,Piero Andrea Temussi,Stephen R. Martin,Giorgio Colombo

doi:10.1371/journal.pone.0095801

Domenico Sanfelice, Annalisa Pastore + Show 5 more

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https://doi.org/10.1371/journal.pone.0095801

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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.

Highlights

Understanding the factors governing the thermal stability of proteins and correlating them with their sequence and structure is a complex and multifaceted problem that can provide important information on the molecular forces involved in protein folding.Frataxins are a family of essential acidic proteins with an a/b fold that is highly conserved both in sequence and structure from bacteria to humans [1]
They play a crucial role in mitochondrial iron metabolism as shown by the fact that neuronal death observed in Friedreich’s ataxia (FRDA, OMIM:229300) patients arises from disregulation of iron homeostasis, with concomitant oxidative damage [1,2]
The thermal stability of Yfh1 in different salts was probed with circular dichroism (CD) spectroscopy by monitoring the intensity at 220 nm in the temperature range 0– 80uC

Summary

Introduction

Frataxins are a family of essential acidic proteins with an a/b fold that is highly conserved both in sequence and structure from bacteria to humans [1]. They play a crucial role in mitochondrial iron metabolism as shown by the fact that neuronal death observed in Friedreich’s ataxia (FRDA, OMIM:229300) patients arises from disregulation of iron homeostasis, with concomitant oxidative damage [1,2]. In agreement with a role in iron metabolism, frataxins bind both Fe(II) and Fe(III) [3,4] but with unexpected if not unique features, which make them unusual proteins. In addition to Fe2+ and Fe3, it is known that other divalent and trivalent cations (Mg2+, Ca2+, Zn2+, Co2+, Al3+ and various lanthanides) [5] bind to the same sites but with lower affinity and specificity

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