Amide proton NMR signals from the N-terminal domain of monomeric a-synuclein (aS) 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 a-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 aS 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. Ca chemical shift data point to <10% residual a-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 1 H-15 N HSQC spectra of aS encapsulated within living Escherichia coli cells. Our finding that temperature effects on aS 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 aS remains predominantly unfolded at physiological temperature and pH-an important conclusion for mechanistic models of the association of aS with membranes and fibrils. Abbreviations: aS, a-synuclein; ct, constant time; CLEANEX, clean chemical exchange; DSS, 2,2-Dimethyl-2-silapentane-5-sulfonic acid; HSQC, heteronuclear single quantum coherence spectroscopy; GM1, monosialotetrahexosylganglioside; R2, transverse relaxation rate (1/ T2); R2 ex , chemical exchange contribution to R2; SDS, sodium dodecyl sulfate.Article and publication are at http://www.proteinscience.org/cgi

The intrinsically unfolded protein a-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK a values for all 26 sites in the protein that ionize below pH 7 using 2D 1 H-15 N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK a values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK a values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For a-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK a of His50, a residue of particular interest since it is flanked within one turn of the a-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK a of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK a shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK a values of uncomplexed a-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the a-synuclein sequence may help nucleate the folding of the protein into an a-helical structure and confer protection from misfolding.

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