Rovere, M. | Search bioRxiv

bioRxiv2018

pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli

N-terminal acetylation is one of the most common post-translational modifications of the eukaryotic proteome and regulates numerous aspects of cellular physiology, such as protein folding, localization and turnover. In particular -synuclein, whose dyshomeostasis has been tied to the pathogenesis of several neurodegenerative disorders, is completely N-acetylated in nervous tissue. In this work, building on previous reports, we develop and characterize a bacterial N-terminal acetylation system based on the expression of the yeast N-terminal acetyltransferase B (NatB) complex under the control of the PBAD (L-arabinose-inducible) promoter. We show its functionality and the ability to completely N-acetylate our model substrate -synuclein both upon induction of the construct with L-arabinose and also by only relying on the constitutive expression of the NatB genes.

molecular biology↗

bioRxiv2017

RECONSTITUTION OF HELICAL SOLUBLE α-SYNUCLEIN THROUGH TRANSIENT INTERACTION WITH LIPID INTERFACES

-synuclein (Syn) is one of the key players in the pathogenesis of Parkinsons disease (PD) and other synucleinopathies. Its misfolding and subsequent aggregation into intracellular inclusions are the pathological hallmark of these diseases and may also play a central role in the molecular cascade leading to neurodegeneration. In this work, we report the existence of a novel soluble -helical conformer of Syn, an archetypal \"intrinsically disordered protein\" (IDP), obtained through transient interaction with lipid interfaces. We describe how the stability of this conformer is highly dependent on the continuous, dynamic oligomerization of the folded species. The conformational space of Syn appears to be highly context-dependent, and lipid bilayers might play crucial roles as molecular chaperones for cytosolic species in a cellular environment, as they do in the case of this previously unreported structure.\n\nSignificance StatementBoth genetic and histopathologic evidence tie -synuclein (Syn) to the pathogenesis of Parkinsons disease (PD), a widespread neurodegenerative disorder. Lipids play a central role in the dynamics of Syn in physiology and disease. Syn undergoes a coil-to-helix transition when binding to lipid vesicles and it is involved in the regulation of synaptic vesicle trafficking. Furthermore, recently discovered -helical, aggregation-resistant \"multimers\" of Syn could constitute a protective conformational pathway. We report the existence of a folded, lipid-unbound Syn conformer that forms upon transient interaction with lipids and is stabilized by dynamic homooligomerization, suggesting that synaptic activity could modulate resistance towards aggregation. Our results are therefore important both for the molecular pathology of PD and the structural biology of intrinsically disordered proteins.

biophysics↗