In-Cell NMR Characterization of the Secondary Structure Populations of a Disordered Conformation of α-Synuclein within E. coli Cells

Christopher A Waudby,John Christodoulou,Anthony W P Fitzpatrick,Lisa D Cabrita,Michele Vendruscolo,Christopher M Dobson,Carlo Camilloni,Piero Andrea Temussi

doi:10.1371/journal.pone.0072286

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.

Highlights

Introduction aSynuclein is a 140-residue protein whose aggregation process is strongly implicated in the pathogenesis of Parkinson’s disease and dementia with Lewy bodies [1,2]
The effect of N-terminal acetylation, a post-translational modification constitutively observed for aSyn in vivo, has been investigated by in-cell NMR for aSyn co-expressed with the N-acetyltransferase NatB within E. coli cells [23]
While small chemical shift changes were observed in the isolated protein following N-terminal acetylation, consistent with the increase in the a-helical population in the first 12 Nterminal residues reported from in vitro studies [24] no additional changes were observed in the HSQC spectrum of the intracellular species [23]

Summary

Introduction

Introduction aSynuclein (aSyn) is a 140-residue protein whose aggregation process is strongly implicated in the pathogenesis of Parkinson’s disease and dementia with Lewy bodies [1,2]. While small chemical shift changes were observed in the isolated protein following N-terminal acetylation, consistent with the increase in the a-helical population in the first 12 Nterminal residues reported from in vitro studies [24] no additional changes were observed in the HSQC spectrum of the intracellular species [23].

Results

Conclusion