Mass spectrometry-based quantitation of methionine oxidation to assess prion structures

Authors: Silva, Christopher - Chris; Erickson-Beltran, Melissa

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/11/2022
Publication Date: 5/20/2022
Citation: Silva, C.J., Erickson-Beltran, M.L. 2022. Mass spectrometry-based quantitation of methionine oxidation to assess prion structures. 10th Iberian Congress on Prions, Vila Real Portugal. May 19-20, 2022. Book Abstract. May 20, 2022. Page 41.

Interpretive Summary:

Technical Abstract: Prions (PrPSc) induce a natively expressed prion protein (PrPC) to adopt the prion conformation. PrPC and PrPSc possess identical primary structures but differ in their 2’, 3’, and quaternary structures. PrPC is a monomer whose tertiary structure has been characterized by NMR and X-ray crystallography. Its secondary structure is composed mostly of disordered motifs, with some a-helical (~29%) and ß-sheet (~2%) motifs. Spectral evidence indicates PrPSc’s secondary structure contains ß-sheet or disordered motifs, with no a-helix. Two structures accommodate these constraints, the parallel-in register intermolecular ß-sheet (PIRIBS) structure and the four-rung ß-solenoid (4RßS) model. The PIRIBS structure is derived from Cryo-EM data. The 4RßS model is a computational model based on constraints imposed by spectral data and steric constraints. A protein’s conformation determines the chemical environment and consequent chemical reactivity of the amino acids in its primary structure. If the same protein can be refolded into more than one conformation, then the same amino acid may react differently with the same added reagent in a conformation-dependent way. Unlike conformation, covalent changes remain after a protein is denatured. In this way conformation-dependent differences can be captured for subsequent analysis. PrPC contains a disproportionately greater number of methionines than other mammalian proteins. Methionines are regarded as a neutral replacement for other non-polar amino acids; in fact, they are labile amino acids that can be oxidized to form a highly polar sulfoxide. Methionine at position 129 in humans and 132 in elk makes them more susceptible to prion diseases. Methionines in the prion conformation are in a different chemical environment and may react differently than the same methionines in the natively expressed prion protein (PrPC) conformation. We tested this hypothesis by reacting the Sc237 strain of hamster-adapted scrapie and recombinant hamster prion protein, which has the same conformation as the natively expressed prion protein, with hydrogen peroxide or chloramine T. The samples were analyzed by mass spectrometry to determine the extent of oxidation of four methionines. As expected, the exposed surface methionines of rPrP were more readily oxidized than those non-surface exposed methionines in the protein’s interior. The same methionines reacted differently when they were contained in the prion conformation. We compared our empirical results with those predicted by the parallel-in register intermolecular ß-sheet (PIRIBS) structure and the four-rung ß-solenoid (4RßS) model. Neither exactly matched our results. However, the 4RßS model more closely matched them than did the PIRIBS structure.