Exploring the surface of the intrinsically disordered protein (IDP) alpha synuclein (AS)

BINOLFI, ANDRES; RIAL HAWILA, ROCÍO; GÓMEZ, GABRIELA; JOSE M. DELFINO

San Luis

Congreso; XLVIII Annual Meeting SAB; 2019

The nature and size of the accessible surface area (SASA) of the polypeptide chain playsa pivotal role in protein folding and complex formation. To investigate SASA, we employdiazirine (DZN), a minute precursor of the extremely reactive methylene carbene (:CH2).Methylation signatures left on the polypeptide provide telltale clues on conformation andinteractions. 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. TheIDP human alpha synuclein (AS) aggregates into oligomers and amyloid fibrils,constituents of Lewy bodies, a cytosolic hallmark of Parkinson disease. DZN labelingproves particularly fit to analyzing the conformational plasticity inherent to this protein.Unlike well-structured proteins where the methylation signal differs strikingly betweennative and unfolded states, AS in buffer or equilibrated in 6 M GdmCl displays a similarlyenhanced EM value, pointing to the high solvent exposure of AS under normalphysiological conditions. Interestingly, compaction of monomeric AS due to calciumbinding yields a somewhat decreased EM value. Most remarkably, AS fibrils cause alarger fall in the EM value, a consequence of surface occlusion at an interface. Trypticfragmentation of AS coupled to MALDI-TOF analysis is revealing methylation patterns atincreased resolution. The peptide coverage achieved permits to build a solventaccessibilitysequence profile. Independently, the feasibility to detect methylatedproducts by multidimensional NMR is an approach that does not demand cleavage of thepolypeptide, opening a potentially rich source of conformational information. The extentof reaction of (:CH2) at various sites across the surface of AS could be defined by 1H15NHSQCspectra. This information illuminates the role played by the constituent parts in themonomeric ensemble as well as the changes observed en route to the fibrillar aggregate.