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.

Low Voltage Voltammetry Probes Proton Dissociation Equilibria of Amino Acids and Peptides.

Platinum-catalyzed electrochemical reduction of dissociable protons at low potentials was used to investigate proton dissociation equilibria of freely diffusing and peptide-incorporated charged amino acids. We first demonstrate with five charged essential amino acids and their analogs that the electrochemically induced deprotonation of each amino acid occurs at distinct formal reduction potential. Moreover, the observed direct reduction for all the charged species, excluding arginine, occurs at low potentials suitable for investigation under aqueous conditions (-0.4 to -0.9 V vs Ag/AgCl). The direct proton reduction was resolved via deconvolution of the observed differential pulse voltammogram (DPV) from background hydronium reduction and water electrolysis. A linear correlation was found between the formal reduction potentials and the pKa values of the dissociable protons hosted by various molecular moieties in the amino acids and their analogs and further verified with tripeptides. DPV of poly(l-lysine) decamer (Lys10) distinctively resolved the pKa values of the amino groups in the side chains and N-terminus, at a resolution not possible by conventional acid-base titration. This work demonstrates selective electrochemical titration of dissociable protons in charged amino acids in the free state and as residues in biomolecules, as well as the utility of DPV to indirectly interrogate local electrostatic environments that are essential to the stability and function of biomolecules.

Unraveling the Influence of Thermal Drawing Parameters on the Microstructure and Thermo-Mechanical Properties of Multimaterial Fibers.

Multimaterial thermally drawn fibers are becoming important building blocks in several foreseen applications in surgical probes, protective gears, or medical textiles. Here, the influence of the thermal drawing parameters on the degree of polymer chain orientation, the related thermal shrinkage behavior, and the mechanical properties of the final fibers is investigated via thermo-mechanical testing and small- and wide-angle X-ray scattering (SAXS and WAXS) analyses. This study on polyetherimide fibers reveals that the drawing stress, which depends on the drawing speed and temperature, controls the thermal shrinkage behavior and mechanical properties. Furthermore, SAXS and WAXS analyses show that the degree of chain orientation increases with drawing stresses below 8 MPa and then saturates, which correlates with the amount of observed shrinkage. The use of this process-dependent polymer chain alignment to tune the mechanical and shrinkage properties of the fibers is highlighted and controlled bending multimaterial fibers made of two polymethyl methacrylates having different molecular weights are developed. Finally, a heat treatment procedure is proposed to relax the chain alignment and increase the dimensional stability of devices such as temperature sensors. This deeper understanding can serve as a guide for the processing of complex fibers requiring specific mechanical properties or enhanced thermal stability.

Voltage-calibrated, finely tunable protein assembly.

Neuronally triggered phosphorylation drives the calibrated and cyclable assembly of the reflectin signal transducing proteins, resulting in their fine tuning of colours reflected from specialized skin cells in squid for camouflage and communication. In close parallel to this physiological behaviour, we demonstrate for the first time that electrochemical reduction of reflectin A1, used as a surrogate for charge neutralization by phosphorylation, triggers voltage-calibrated, proportional and cyclable control of the size of the protein's assembly. Electrochemically triggered condensation, folding and assembly were simultaneously analysed using in situ dynamic light scattering, circular dichroism and UV absorbance spectroscopies. The correlation of assembly size with applied potential is probably linked to reflectin's mechanism of dynamic arrest, which is controlled by the extent of neuronally triggered charge neutralization and the corresponding fine tuning of colour in the biological system. This work opens a new perspective on electrically controlling and simultaneously observing reflectin assembly and, more broadly, provides access to manipulate, observe and electrokinetically control the formation of intermediates and conformational dynamics of macromolecular systems.

Reversible electrochemical triggering and optical interrogation of polylysine α-helix formation.

Reversible electrochemical triggering of the random coil to α-helix conformational transition of polylysine (Lys10, Lys20, Lys50) was accomplished at a Pt electrode at potentials < |1| V vs. Ag/AgCl. Direct electroreduction of the N-terminus vs ε-amino groups in Lys sidechains, as well as hydronium reduction and electrolysis, could be easily distinguished and deconvolved using differential pulse voltammetry. Electrochemistry was coupled with in situ UV absorbance and circular dichroism spectroscopies to dynamically follow the evolution of α-helix formation at different potentials. Isotope experiments in H2O vs. D2O unequivocally confirm that direct electroreduction of ε-NH3+/ND3+ groups in Lys sidechains, rather than electrochemically generated pH gradient-induced deprotonation, leads to subsequent α-helix formation. The site-selective electrochemistry and optical methodologies presented herein can be generalized and extended to interrogate other protonation-sensitive biomolecular systems, and potentially provide access to early intermediates and control over the dynamic structural evolution of peptides and proteins.