In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-synuclein within E. coli cells

Abstract

α-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson's disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, α-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution.

Figure 1. Multidimensional deconvolution of in-cell NMR spectra.

HNCO spectra of αSyn (A) expressed within E. coli cells and (B) following deconvolution using the T92 reference peak indicated in (A). (C,D) ¹H/¹⁵N projections of (C) the original HNCO spectrum and (D) the spectrum following deconvolution.

Figure 2. Analysis of backbone chemical shift changes and line broadening.

(A) Overlay of ¹³C/¹⁵N slices through the HNCO spectrum of αSyn in bulk solution (green) and the deconvolved spectrum of αSyn expressed within cells (blue), showing resonances with ¹H chemical shifts between 8.45 and 8.55 ppm. (B–F) Backbone chemical shift changes observed for intracellular αSyn relative to the protein in bulk solution. (G) Relative HNCO intensities of intracellular αSyn compared to αSyn in bulk aqueous solution.

Figure 3. Comparison of secondary structure populations in isolated and intracellular αSyn.

Secondary structure populations of αSyn calculated with the δ2D method using the backbone chemical shifts of (A) intracellular αSyn, (B) monomeric αSyn in bulk solution at the same pH, and (C) SDS micelle-bound αSyn at pH 7.4. (D) Differences in secondary structure populations between intracellular and bulk solution measurements.