A new electrochemical method that mimics phosphorylation of the core tau peptide K18 enables kinetic and structural analysis of intermediates and assembly.

Tau protein's reversible assembly and binding of microtubules in brain neurons are regulated by charge-neutralizing phosphorylation, while its hyperphosphorylation drives the irreversible formation of cytotoxic filaments associated with neurodegenerative diseases. However, the structural changes that facilitate these diverse functions are unclear. Here, we analyzed K18, a core peptide of tau, using newly developed spectroelectrochemical instrumentation that enables electroreduction as a surrogate for charge neutralization by phosphorylation, with simultaneous, real-time quantitative analyses of the resulting conformational transitions and assembly. We observed a tipping point between behaviors that paralleled the transition between tau's physiologically required, reversible folding and assembly and the irreversibility of assemblies. The resulting rapidly electroassembled structures represent the first fibrillar tangles of K18 that have been formed in vitro at room temperature without using heparin or other charge-complementary anionic partners. These methods make it possible to (i) trigger and analyze in real time the early stages of conformational transitions and assembly without the need for preformed seeds, heterogenous coacervation, or crowding; (ii) kinetically resolve and potentially isolate never-before-seen early intermediates in these processes; and (iii) develop assays for additional factors and mechanisms that can direct the trajectory of assembly from physiologically benign and reversible to potentially pathological and irreversible structures. We anticipate wide applicability of these methods to other amyloidogenic systems and beyond.

Kinetics aspects of Gamma-hydroxybutyrate dehydrogenase.

Two groups of metabolically related enzymes, the Group III family of Fe2+-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD+ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe2+ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.