X-ray Absorption Spectroscopy Reveals a Substantial Increase of Sulfur Oxidation in Transthyretin (TTR) upon Fibrillization

Abstract

Transthyretin (TTR) amyloid fibrils are the main component of the amyloid deposits occurring in Familial Amyloidotic Polyneuropathy patients. This is 1 of 20 human proteins leading to protein aggregation disorders such as Alzheimer's and Creutzfeldt-Jakob diseases. The structural details concerning the association of the protein molecules are essential for a better understanding of the disease and consequently the design of new strategies for diagnosis and therapeutics. Disulfide bonds are frequently considered essential for the stability of protein aggregates and since in the TTR monomers there is one cysteine residue, it is important to determine unambiguously the redox state of sulfur present in the fibrils. In this work we used x-ray spectroscopy to further characterize TTR amyloid fibrils. The sulfur K-edge absorption spectra for the wild type and some amyloidogenic TTR variants in the soluble and fibrillar forms were analyzed. Whereas in the soluble proteins the thiol group from cysteine (R-SH) and the thioether group from methionine (R-S-CH 3 ) are the most abundant forms, in the TTR fibrils there is a significant oxidation of sulfur to the sulfonate form in the cysteine residue and a partial oxidation of sulfur to sulfoxide in the methionine residues. Further interpretation of the data reveals that there are no disulfide bridges in the fibrillar samples and suggest conformational changes in the TTR molecule, namely in strand A and/or in its vicinity, upon fibril formation.

Human transthyretin in complex with iododiflunisal: structural features associated with a potent amyloid inhibitor

“…Interestingly, the crystal structure of the highly amyloidogenic L55P-TTR variant revealed a structural change of the edge strand D, leading to a long loop between strands C and E [10]. Furthermore, the displacement of strand D is in agreement with the observation that the protein's sulphur-containing amino acids became oxidized on fibrillization [11], since these residues are in the vicinity of the displaced strand and probably become more accessible to the solvent.…”

Towards a Structural Understanding of the Fibrillization Pathway in Machado-Joseph's Disease: Trapping Early Oligomers of Non-expanded Ataxin-3

“…59 Briefly, ThT (final concentration 30 mM) was added to 100 mg of protein in 50 mM glycine/NaOH buffer (pH 9.0), in an assay volume of 1 ml. Excitation and emission slits were set at 5 nm and 10 nm, respectively.…”

Packing of the Prion Ure2p in Protein Fibrils Probed by Fluorescence X-ray Near-edge Structure Spectroscopy at Sulfur K-edge

“…In particular, XANES at the sulfur K-edge allows us to discriminate and quantify the ratio of reduced forms versus oxidized forms of sulfur-containing amino acids in proteins. 20 Ure2p contains nine methionine residues per monomer and is devoid of cysteine residues. The dimeric form of the protein is in equilibrium with a monomeric state.…”