Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies

Abstract

Thermodynamic and pH stability of recombinant human L- and H-ferritins were probed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7. At pH 2.0-2.8 they were dissociated into subunit monomers and in this pH interval the H-subunit displayed a single calorimetrically-revealed domain with properties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or 169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), a wide transition range (approximately 20 degrees C) and high ANS binding. In contrast, at pH 2 the L-ferritin subunit showed two calorimetric domains with Tm of 35 and 40 degrees C with similar unfolding enthalpies and with moderate extent of interactions, as indicated by the ratio of calorimetric enthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for the thermal transition. A pH increase from 2.0 to 2.8 determined the coupling of the two domains into a single cooperative folding unit and drastic increase of the transition temperature (from 37 to 80 degrees C). The contacts between the two domains in the L-subunit appeared to contribute to about 30% of the total stabilization free energy. The unfolding enthalpies, heat capacity changes and pronounced ANS binding of the L-subunit at pH 2.0-2.8 indicated that part of the structure lacked 'meltable' tertiary interactions. The results indicate that H- and L-subunits are stabilized by largely different intra-chain interactions with a critical contribution to L-subunit stability of embedded salt bridge(s) absent in the H-subunit.

Iron-mediated Aggregation and a Localized Structural Change Characterize Ferritin from a Mutant Light Chain Polypeptide That Causes Neurodegeneration

“…We found that in the presence of 4.5 M GdnHCl, WT-FTL homopolymers denatured with the higher T m ϳ90°C and MT-FTL at ϳ45°C. Without addition of the destabilizing agent, MT-FTL denatured near 75°C, which is reduced considerably from that reported in the literature (30,32) for the wild type, and again points toward significant destabilization of the mutant. For comparison, FTH1 homopolymers are less stable to heat denaturation than FTL homopolymers, perhaps because of residues located at the intersubunit contacts along the 3-and 4-fold channels and by salt bridges within the 4-helix bundles themselves between Lys-62 and Glu-107 (24,32).…”

“…Both wild type FTH1 and FTL polypeptide subunits are important for the iron storage function of ferritin, with the former containing the ferroxidase site for ferrous iron oxidation and the latter containing the iron nucleation site (2, 3). However, homopolymers composed of FTL subunits have additional properties such as resistance to precipitation under iron loading and being significantly more stable to denaturation by heat and solvent (30,36), and apparently FTL subunits are able to confer these properties upon the heteropolymer. Thus as the first approach to elucidating the effects of the mutation, we compared and contrasted wild type with MT-FTL homopolymers, which have a C terminus of altered length and composition (9).…”

“…alfold, and the emission maximum is blue-shifted to 470 nm. At pH 7.4, native ferritin does not bind ANS nor does it fully denatured ferritin (24,30). However, a large increase in ANS fluorescence at 470 nm was observed when ANS was added to the MT-FTL homopolymers, indicating exposed hydrophobic sites (Fig.…”

Biology of ferritin in mammals: an update on iron storage, oxidative damage and neurodegeneration

“…The 24 subunits come together in a three-dimensional structure that is designed for the dynamic storage of iron; extensive intra-and inter-subunits helix-helix interactions give rise to a very stable protein (it resists up to 80C°, 6M guanidine and pH 3-10) (Martsev et al 1998) yet maintaining sufficient flexibility to allow the flux of iron and other small molecules in and out of its shell (Liu and Theil 2005). The protein actually shows eight narrow hydrophilic channels around the 3-fold symmetry axes and six hydrophobic channels around the 4-fold axes, all with a diameter of about 0.4 nm (figure 1).…”

Ferritin and ferritin isoforms I: Structure–function relationships, synthesis, degradation and secretion

“…The contributions of these types of interactions to the folding and stability of the H-and L-subunits differ significantly between the two types of subunits. The intrasubunit hydrogen bonds are about 50% more abundant in the H-subunit than in the L-subunit, whereas the salt bridges are more important in the stabilization of the L-subunit and accounts for about 30% of the stabilization energy of the L-subunit (Martsev et al, 1998). Due to the large number of intra-and inter-subunit salt bridges the ferritin molecule is highly stable to thermal and chemical denaturation (Harrison & Arosio, 1996).…”