NMR determination of pKa values in α-synuclein.

The intrinsically unfolded protein α-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 α-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 α-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 α-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 α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding.

Electrostatic contributions to the stabilities of native proteins and amyloid complexes.

The ability to predict electrostatic contributions to protein stability from structure has been a long-standing goal of experimentalists and theorists. With recent advances in NMR spectroscopy, it is possible to determine pK(a) values of all ionizable residues for at least small proteins, and to use the pK(a) shift between the folded and unfolded states to calculate the thermodynamic contribution from a change in charge to the change in free energy of unfolding. Results for globular proteins and for α-helical coiled coils show that electrostatic contributions to stability are typically small on an individual basis, particularly for surface-exposed residues. We discuss why NMR often suggests smaller electrostatic contributions to stability than X-ray crystallography or site-directed mutagenesis, and discuss the type of information needed to improve structure-based modeling of electrostatic forces. Large pK(a) shifts from random coil values are observed for proteins bound to negatively charged sodium dodecyl sulfate micelles. The results suggest that electrostatic interactions between proteins and charges on the surfaces of membrane lipid bilayers could be a major driving force in stabilizing the structures of peripheral membrane proteins. Finally, we discuss how changes in ionization states affect amyloid-ß fibril formation and suggest that electrostatic repulsion may be a common destabilizing force in amyloid fibrils.

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.