Conformational Properties of the SDS-Bound State of α-Synuclein Probed by Limited Proteolysis:  Unexpected Rigidity of the Acidic C-Terminal Tail

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α-Synuclein (α-syn) is a “natively unfolded” protein constituting the major component of intracellular inclusions in several neurodegenerative disorders. Here, we describe proteolysis experiments conducted on human α-syn in the presence of SDS micelles. Our aim was to unravel molecular features of micelle-bound α-syn using the limited proteolysis approach. The nonspecific proteases thermolysin and proteinase K, as well as the Glu-specific V8-protease, were used as proteolytic probes. While α-syn at neutral pH is easily degraded to a variety of relatively small fragments, in the presence of 10 mM SDS the proteolysis of the protein is rather selective. Complementary fragments 1−111 and 112−140, 1−113 and 114−140, and 1−123 and 124−140 are obtained when thermolysin, proteinase K, and V8 protease, respectively, are used. These results are in line with a conformational model of α-syn in which it acquires a folded helical structure in the N-terminal region in its membrane-bound state. At the same time, they indicate that the C-terminal portion of the molecule is rather rigid, as seen in its relative resistance to extensive proteolytic degradation. It is likely that, under the specific experimental conditions of proteolysis in the presence of SDS, the negatively charged C-terminal region can be rigidified by binding a calcium ion, as shown before with intact α-syn. In this study, some evidence of calcium binding properties of isolated C-terminal fragments 112−140, 114−140, and 124−140 was obtained by mass spectrometry measurements, since molecular masses for calcium-loaded fragments were obtained. Our results indicate that the C-terminal portion of the membrane-bound α-syn is quite rigid and structured, at variance from current models of the membrane-bound protein deduced mostly from NMR. Considering that the aggregation process of α-syn is modulated by its C-terminal tail, the results of this study may provide useful insights into the behavior of α-syn in a membrane-mimetic environment.

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Biochemistry, v. 45, issue 38, p. 11523-11531