Research: [ CITATION Ann13 \/ 2057 ] - Disruption of FeS cluster biosynth, mitochondrial overload, oxidative stress. - Frn: highly conserved iron binding protein. Crucial element in FeS cluster biogenesis. - FA caused by abnormal expansion of non-coding GAA triplet repeat in the first intron of the frn gene -> lower exp of frn through heterochromatization of the locus. - Frn: small acidic protein. Highly conserved in most organisms- bacteria to mammals. - Sequence alignment of the frn family shows 2 distinct regions: Nterm block of 70-90 residues - completely absent in prokaryotes and poorly conversed among eukaryotes. - C term: highly conserved block of 100-200 amino acids- conserved in most organisms. - Sequence identity = 25%, similarity = 40% - functionally important part of that molecule. - Frn is imported into mitochondria through import signal contained I N term: absence of non- conserved Nterm in prokaryotes and homologs in purple bacterial genomes but not others = mitochondrial localisation. - Tagging expts showed frn co-localises with mitochondrial protein. - Frn is synth as precursor of 210 aas imported to MT- matures by MT processing peptidase leads to gen on an intermediate form of 19kDa cleaved between G41 and L42 and a mature form of 14kDa- fully functional for cell survival. 3D structure of Frn: - Structure of conserved C term domain of the human protein: first to be characterized by NMR and crystallography. - Solution structures of bacterial frn and of Yfh1 in crystal and solution states solved later: superpose with RMSD of 1.7=2.4 angstroms in agreement with high degree of sequence conservation. - Two N and C term regions. - Nterm eukaryotic tail of human protein (res 81-92): intrinsically unfolded and highly flexible. Equiv. in YfH1 is more rigid. - In xray structure: region is visible and involved in intermolecular interactions with other copies of molecule. - in sol structure: bends back in conformation that is shared by all copies of the NMR bundle - but should adopt diff conformations in diff models if it were flexible. - Conserved C term domain: folds in a mixed alpha-beta structure that consists of 2 helices packing against a contiguous anti-parallel beta-sheets assembled in the sequence alpha- (beta)5-7alpha. - Bacterial, yeast and human frn have the same fold but diff thermodynamic stabilities. - Yfh1: unstable when studied at low ionic strength. Midpoint of Tm is 35 degrees c. - The length of the c term domain is variable in the 3 proteins - affects stability. - Human frn: c term region folds back and inserts between the 2 helices- protecting the h.phobic core. - E.coli: shorter c term. - In the frn family, the semi-conserved negatively charged residues and other exposed conserved residues cluster on the first helix and on the beta sheet. - Among these residues are well-exposed and conserved aspartate and tryptophan. - All 3 orthologs studied are highly soluble monomeric proteins in he absence of iron.
- When iron excesses are added under aerobic conditions oligomers are observed for