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

Amide proton NMR signals from the N-terminal domain of monomeric alpha-synuclein (alphaS) are lost when the sample temperature is raised from 10 degrees C to 35 degrees C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in alpha-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 alphaS 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. Calpha chemical shift data point to <10% residual alpha-helix structure at 10 degrees C and 35 degrees 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 degrees C in 1H-15N HSQC spectra of alphaS encapsulated within living Escherichia coli cells. Our finding that temperature effects on alphaS 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 alphaS remains predominantly unfolded at physiological temperature and pH-an important conclusion for mechanistic models of the association of alphaS with membranes and fibrils.