Fibrillation of the intrinsically disordered protein (IDP) alpha synuclein (AS) probed by surface methylation.

RIAL HAWILA R; GÓMEZ GE; BINOLFI A; ROSANO G; DELFINO JM

Rosario, Santa Fe, Argentina.

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica (SAB).; 2022

Sociedad Argentina de Biofísica (SAB)

Fibrillation of the intrinsically disordered protein (IDP) alpha synuclein(AS) probed by surface methylationRial Hawila Ra, Gómez GEa, Binolfi Ab, Rosano Gb, Delfino JMaa - Departamento de Química Biológica e IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica,Universidad de Buenos Airesb - Instituto de Biología Molecular y Celular de Rosario IBR (UNR-CONICET), Rosario, Argentina.The nature and size of the accessible surface area (SASA) of the polypeptide chain plays akey role in protein folding and complex formation. SASA can be probed with diazirine(DZN), a tiny precursor of the extremely reactive methylene carbene (:CH2). Sparsemethylated sites left on the polypeptide provide revealing signs on the conformation. Themetric extent of methylation (EM) derived from mass spectra discriminates betweennative and alternate states. Human alpha synuclein (AS) aggregates into oligomers andamyloid fibrils, constituents of Lewy bodies, a cytosolic hallmark of Parkinson disease. DZNlabeling proves particularly fit to analyzing the conformational plasticity inherent to thisintrinsically disordered protein. Unlike folded proteins where EM differs markedly betweennative and unfolded states, AS in buffer or in 6 M GdmCl displays a similarly enhancedvalue, revealing high solvent accessibility under normal physiological conditions.Strikingly, by using ESI-MS it was shown that AS fibrils display an enhanced globalmethylation compared to the monomer and also, incorporating the higher accuracy andresolution technology of the MS-Orbitrap we were able to see methylation at theindividual amino acid level. In addition, location of methylated sites by multidimensionalNMR (1H15N-HSQC) opens a vast panorama. The extent of :CH2 reaction across the surfaceof AS defines sequential intensity profiles. Differential labeling signs point to theinvolvement of a particular region in the NAC domain (75-88) in fibril formation as aconsequence of the organization of a hydrophobic interface. We anticipate that thecombined picture derived from MS and NMR data will illustrate characteristic signaturesfor the molecular species involved.AcknowldegmentsUBACyT, CONICET and ANPCyT176