Conformational Properties of the SDS-Bound State of -Synuclein Probed by Limited Proteolysis: Unexpected Rigidity of the Acidic C-Te
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- 作者：Patrizia Polverino de Laureto ; Laura Tosatto ; Erica Frare ; Oriano Marin ; Vladimir N. Uversky ; Angelo Fontana
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：September 26, 2006
- 年：2006
- 卷：45
- 期：38
- 页码：11523 - 11531
- 全文大小：305K
- 年卷期：v.45,no.38(September 26, 2006)
- ISSN：1520-4995
文摘
-Synuclein (-syn) is a "natively unfolded" protein constituting the major component ofintracellular inclusions in several neurodegenerative disorders. Here, we describe proteolysis experimentsconducted on human -syn in the presence of SDS micelles. Our aim was to unravel molecular featuresof micelle-bound -syn using the limited proteolysis approach. The nonspecific proteases thermolysinand proteinase K, as well as the Glu-specific V8-protease, were used as proteolytic probes. While -synat neutral pH is easily degraded to a variety of relatively small fragments, in the presence of 10 mM SDSthe proteolysis of the protein is rather selective. Complementary fragments 1-111 and 112-140, 1-113and 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 -syn in which it acquiresa folded helical structure in the N-terminal region in its membrane-bound state. At the same time, theyindicate that the C-terminal portion of the molecule is rather rigid, as seen in its relative resistance toextensive proteolytic degradation. It is likely that, under the specific experimental conditions of proteolysisin the presence of SDS, the negatively charged C-terminal region can be rigidified by binding a calciumion, as shown before with intact -syn. In this study, some evidence of calcium binding properties ofisolated C-terminal fragments 112-140, 114-140, and 124-140 was obtained by mass spectrometrymeasurements, since molecular masses for calcium-loaded fragments were obtained. Our results indicatethat the C-terminal portion of the membrane-bound -syn is quite rigid and structured, at variance fromcurrent models of the membrane-bound protein deduced mostly from NMR. Considering that theaggregation process of -syn is modulated by its C-terminal tail, the results of this study may provideuseful insights into the behavior of -syn in a membrane-mimetic environment.