GOMEZ Gabriela Elena
congresos y reuniones científicas
Exploring the surface of the intrinsically disordered protein (IDP) alpha synuclein (AS).
RIAL HAWILA, MR; GÓMEZ, GABRIELA E; BINOLFI A; DELFINO, J.M
Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019
The nature and size of the accessible surface area (SASA) of the polypeptide chain plays a pivotal role in protein folding and complex formation. To investigate SASA, we employ diazirine (DZN), a minute precursor of the extremely reactive methylene carbene (:CH2). Methylation signatures left on the polypeptide provide telltale clues on conformation and interactions. The extent of methylation metric (EM) derived directly from mass spectra (ESI-MS or MALDI-TOF) is able to discriminate between native and alternate states. The IDP human alpha synuclein (AS) aggregates into oligomers and amyloid fibrils, constituents of Lewy bodies, a cytosolic hallmark of Parkinson disease. DZN labeling proves particularly fit to analyzing the conformational plasticity inherent to this protein. Unlike well-structured proteins where the methylation signal differs strikingly between native and unfolded states, AS in buffer or equilibrated in 6 M GdmCl displays a similarly enhanced EM value, pointing to the high solvent exposure of AS under normal physiological conditions. Interestingly, compaction of monomeric AS due to calcium binding yields a somewhat decreased EM value. Most remarkably, AS fibrils cause a larger fall in the EM value, a consequence of surface occlusion at an interface. Tryptic fragmentation of AS coupled to MALDI-TOF analysis is revealing methylation patterns at increased resolution. The peptide coverage achieved permits to build a solvent-accessibility sequence profile. Independently, the feasibility to detect methylated products by multidimensional NMR is an approach that does not demand cleavage of the polypeptide, opening a potentially rich source of conformational information. The extent of reaction of (:CH2) at various sites across the surface of AS could be defined by 1H15N-HSQC spectra. This information illuminates the role played by the constituent parts in the monomeric ensemble as well as the changes observed en route to the fibrillar aggregate.