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This Article Full Text Full Text (PDF) All Versions of this Article: ps.033803.107v1 17/8/1434    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Croke, R. L. Articles by Alexandrescu, A. T. PubMed PubMed Citation Articles by Croke, R. L. Articles by Alexandrescu, A. T. Social Bookmarking                   What's this?

Hydrogen exchange of monomeric -synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

¹ Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125, USA
² Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA

(RECEIVED December 3, 2007; FINAL REVISION May 7, 2008; ACCEPTED May 12, 2008)

Amide proton NMR signals from the N-terminal domain of monomeric -synuclein (S) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in -helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of S than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. C chemical shift data point to <10% residual -helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of S encapsulated within living Escherichia coli cells. Our finding that temperature effects on S NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that S remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of S with membranes and fibrils.

Keywords: Parkinson's disease; intrinsically unfolded proteins; amyloid; exchange broadening; in-cell NMR; membrane proteins

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