Denaturation of horse spleen ferritin in aqueous guanidinium chloride solutions

Listowsky, Irving; Blauer, G.; Enlard, S; Jj, Betheil

doi:10.1021/bi00761a026

Cited by 77 publications

(55 citation statements)

References 26 publications

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“…b Transfection control is plasmid with no insert; HuFtH-WT is plasmid encoding human wild type ferritin H; HuFtH L138P is plasmid encoding human ferritin H with pores unfolded by substitution of a conserved amino acid in the pore/channel. c Data are significantly different from pore mutant (HuFtH L138P) or transfection control: ***, p Ͻ 0.005, and *, p Ͻ 0.05. have no effect on the overall protein cage structure, based on CD spectroscopy and gel filtration (14,34,35). Ferritin pores are formed by six helices contributed by three subunits near the 3-fold symmetry axis of the protein cage, placing each pore roughly at one of the eight corners of a cube centered on the protein cage.…”

Section: Discussionmentioning

confidence: 99%

Ferritin Contains Less Iron (59Fe) in Cells When the Protein Pores Are Unfolded by Mutation

Hasan¹,

Tosha

Theil

2008

Journal of Biological Chemistry

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Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe 2؉ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mM) and monitored by CD spectroscopy, protein crystallography, and Fe 2؉ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t1 ⁄ 2 decreased 17-fold), and (iii) decreased ␣-helix (8%). Overexpression (>4-fold), in

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Section: Discussionmentioning

confidence: 99%

Ferritin Contains Less Iron (59Fe) in Cells When the Protein Pores Are Unfolded by Mutation

Hasan¹,

Tosha

Theil

2008

Journal of Biological Chemistry

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“…No dissociation of cage subunits or helix unfolding is observed in 6 M urea or 6 M guanidine unless the pH is less than 3.5 [27]. In addition, using changes in the absorbance at 280 nm to monitor global changes in cage folding, nanocage structure was stable to 85 °C in both wild type ferritin and ferritin with pore mutations [3] (Fig.…”

Section: Selective Unfolding or Melting Of Ferritin Protein Nanocage mentioning

confidence: 94%

Gated pores in the ferritin protein nanocage

Theil

Liu

Tosha

2008

Inorganica Chimica Acta

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Synopsis and pictogram: Gated pores in the ferritin family of protein nanocages, illustrated in the pictogram, control transfer of ferrous iron into and out of the cages by regulating contact between hydrated ferric oxide mineral inside the protein cage, and reductants such as FMNH 2 on the outside. The structural and functional homology between the gated ion channel proteins in inaccessible membranes and gated ferritin pores in the stable, water soluble nanoprotein, make studies of ferritin pores models for gated pores in many ion channel proteins.Properties of ferritin gated pores, which control rates of FMNH 2 reduction of ferric iron in hydrated oxide minerals inside the protein nanocage, are discussed in terms of the conserved pore gate residues (arginine 72-apspartate 122 and leucine 110-leucine 134), of pore sensitivity to heat at temperatures 30 °C below that of the nanocage itself, and of pore sensitivity to physiological changes in urea (1-10 mM). Conditions which alter ferritin pore structure/function in solution, coupled with the high evolutionary conservation of the pore gates, suggest the presence of molecular regulators in vivo that recognize the pore gates and hold them either closed or open, depending on biological iron need. The apparent homology between ferrous ion transport through gated pores in the ferritin nanocage and ion transport through gated pores in ion channel proteins embedded in cell membranes, make studies of water soluble ferritin and the pore gating folding/unfolding a useful model for other gated pores.

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“…However, the optical rotatory properties changed in presence of 6 M guanidine hydrochloride at pH 6.0 consistent with helix disruption in the protein. [185,188,223,224] By far the most useful procedure for the reversible dissociation of apoferritin is that described by P. M. Harrison and D. W. Gregory in 1965 in which they observed the disassembly of the protein shell into its subunits as a function of pH. [225] In 1968, they also showed that apoferritin subunits are able to assemble in the absence of iron as determined by electrophoretic mobility and sedimentation coefficient analyses, which led to the conclusion that the reassembled apoferritin is identical to the native ferritin.…”

Section: Electrostatic Featuresmentioning

confidence: 99%