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

Abstract

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.

Figure 1.

NMR spectra of αS at 10°C (A–C) and 35°C (D–F). The panels show ¹H-¹⁵N HSQC spectra (A,D), portions of Hα–Cα selective ct-¹H-¹³C HSQC spectra (B,E), and aromatic-selective ct-¹H-¹³C HSQC spectra (C,F). Samples were in 20 mM sodium phosphate, pH 7.4. Boxed correlations have negative phases in the ct-¹H-¹³C HSQC spectra. Residue type assignments are indicated by single-letter amino acid codes. Because of crowding in A we labeled only three regions of the spectrum that contain the majority of glycine, threonine, and alanine correlations, respectively. A complete set of sequence-specific assignments for the spectrum in A is provided in Supplemental Figure S1. In D, sequence-specific assignments are given for the subset of cross peaks that remain at 35°C. The assignments in D were obtained by following chemical shifts changes in ¹H-¹⁵N HSQC spectra at 5° intervals between 35°C and 10°C. Most peaks that survive at 35°C are from slowly exchanging residues in the C-terminal domain (residues 100–140). Some weaker correlations are also seen for valines 16, 37, 48, 71, and 74 from the N-terminal domain, since the valines have slow intrinsic exchange rates. Superscripts in B, C, E, and F denote carbon types (e.g., Y^δ are the delta carbons of the tyrosine ring). Labels without superscripts in panels B and E are Hα–Cα cross peaks.

Figure 2.

NMR spectra of αS without (A–C) or with 45 mM SDS micelles (D–F). All spectra were obtained at 10°C, pH 7.4. The panels show ¹H-¹⁵N HSQC spectra (A,D), Hα–Cα selective ct-¹H-¹³C HSQC spectra (B,E), and aromatic-selective ct-¹H-¹³C HSQC spectra (C,F). Boxed correlations have negative phases in the ct-¹H-¹³C HSQC spectra. A complete set of sequence-specific assignments for the spectrum shown in A is provided in Supplemental Figure S1. Sequence-specific assignments in B are for all cross peaks that remain with 45 mM SDS.

Figure 3.

Amide hydrogen exchange rates of αS. (A) Theoretically predicted intrinsic exchange rates (Bai et al. 1993) for αS at pH 7.4 and a temperature of 15°C. The rates were calculated with the SPHERE server (Zhang 1995). (B) Experimentally determined amide proton exchange rates from CLEANEX spectroscopy (Hwang et al. 1998) using an αS sample at low salt (20 mM sodium phosphate). (C) Hydrogen exchange rates as in B, but for a sample containing 20 mM phosphate and 300 mM NaCl. Only well-resolved HN correlations were included in the analysis. Uncertainties in exchange rates were on the order of ∼20% of the values for the low salt sample, and 40% for the high salt sample. Rates and uncertainties are given in Supplemental Table 1.

Figure 4.

Recovery of αS ¹H-¹⁵N correlations with decreasing pH (A–D) and αS in live E. coli cells (E). Spectra in A–D are of purified αS in 20 mM phosphate buffer at a temperature of 35°C and the indicated pH values. The spectrum in E is of αS within living E. coli cells and was obtained at a temperature of 37°C. The cross peak of Ala140 denoted by “f(A140)” is aliased in the ¹⁵N dimension in A–D, but is outside of the region shown in E. Arrows in E indicate cross peaks from unknown E. coli metabolites (Serber et al. 2001). While these peaks fall within the random coil region of the ¹H-¹⁵N HSQC spectrum, their intensities are usually smaller than the αS signals.

Figure 5.

Differences in Cα chemical shifts from random coil values (Wishart et al. 1995a). Black circles, monomeric αS at pH 6.4 and a temperature of 10°C; gray triangles, monomeric αS at pH 6.4 and a temperature of 35°C; squares with dotted lines, micelle-bound αS (Chandra et al. 2003).