The Factors Governing the Thermal Stability of Frataxin Orthologues:  How To Increase a Protein's Stability

Abstract

Understanding the factors governing the thermal stability of proteins and correlating them to the sequence and structure is a complex and multiple problem that can nevertheless provide important information on the molecular forces involved in protein folding. Here, we have carried out a comparative genomic study to analyze the effects that different intrinsic and environmental factors have on the thermal stability of frataxins, a family of small mitochondrial iron-binding proteins found in organisms ranging from bacteria to humans. Low expression of frataxin in humans causes Friedreich's ataxia, an autosomal recessive neurodegenerative disease. The human, yeast, and bacterial orthologues were selected as representatives of different evolutionary steps. Although sharing high sequence homology and the same three-dimensional fold, the three proteins have a large variability in their thermal stabilities. Whereas bacterial and human frataxins are thermally stable, well-behaved proteins, under the same conditions yeast frataxin exists in solution as an unstable species with apprechable tracts in a conformational exchange. By designing suitable mutants, we show and justify structurally that the length of the C-terminus is an important intrinsic factor that directly correlates with the thermal stabilities of the three proteins. Thermal stability is also gained by the addition of Fe(2+). This effect, however, is not uniform for the three orthologues nor highly specific for iron: a similar albeit weaker stabilization is observed with other mono- and divalent cations. We discuss the implications that our findings have for the role of frataxins as iron-binding proteins.

Yeast Frataxin Solution Structure, Iron Binding, and Ferrochelatase Interaction,

“…Using a 15 N-labeled frataxin concentration of 333 μM, aqueous anaerobic ferrous ammonium sulfate (Sigma) in NMR buffer was added to independent protein samples to achieve Fe(II):protein stoichiometric ratios of 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, and 2.00. 15 N-labeled frataxin in NMR buffer with 650 μM ammonium sulfate (Sigma) served as our apoprotein control. Samples were prepared at room temperature immediately before data collection, placed in the NMR spectrometer, and allowed to equilibrate to 30 °C for 30 min.…”

“…The protein complex sample was prepared in NMR buffer with 0.005% deoxycholate, at a stoichiometric monomer ratio of 1:1 at a final individual protein concentration of 300 μM. 15 N-labeled frataxin in NMR buffer with 0.005% deoxycholate served as our frataxin control. Because of the large size of the protein complex (yeast ferrochelatase is a dimer at ca.…”

“…Nearly complete resonance and β-methylene stereospecific assignments were made for frataxin using 1 H, 13 C, 15 N heteronuclear experiments. There is no indication within our data that there is more than a single protein conformation in solution, and our protein behaves like a well-folded homogeneous molecule for multiple weeks when NMR samples are stored under argon in sealed tubes (15).…”

“…Nearly complete resonance and β-methylene stereospecific assignments were made for frataxin using 1 H, 13 C, 15 N heteronuclear experiments. There is no indication within our data that there is more than a single protein conformation in solution, and our protein behaves like a well-folded homogeneous molecule for multiple weeks when NMR samples are stored under argon in sealed tubes (15). A total of 1730 NOE distance restraints, combined with 144 dihedral angle restraints (obtained from TALOS), 44 hydrogen bonding restraints (based on NOE patterns and chemical shift index analysis), and 80 H β stereospecific assignments (based on NOE patterns), were used to calculate 200 independent structures with the torsion angle dynamics simulated annealing algorithm within DYANA.…”

“…The presence of iron can induce aggregation in human and yeast frataxins (12,13); however, this oligomerization is dependent on solution conditions (11,15). Monomeric human and bacterial frataxins have been reported to bind up to six iron atoms, with dissociation constants of 55 and 3.8 μM, respectively (4,14).…”