Electrochemistry as a surrogate for protein phosphorylation: voltage-controlled assembly of reflectin A1

Abstract

Phosphorylation is among the most widely distributed mechanisms regulating the tunable structure and function of proteins in response to neuronal, hormonal and environmental signals. We demonstrate here that the low-voltage electrochemical reduction of histidine residues in reflectin A1, a protein that mediates the neuronal fine-tuning of colour reflected from skin cells for camouflage and communication in squids, acts as an in vitro surrogate for phosphorylation in vivo , driving the assembly previously shown to regulate its function. Using micro-drop voltammetry and a newly designed electrochemical cell integrated with an instrument measuring dynamic light scattering, we demonstrate selective reduction of the imidazolium side chains of histidine in monomers, oligopeptides and this complex protein in solution. The formal reduction potential of imidazolium proves readily distinguishable from those of hydronium and primary amines, allowing unequivocal confirmation of the direct and energetically selective deprotonation of histidine in the protein. The resulting ‘electro-assembly’ provides a new approach to probe, understand, and control the mechanisms that dynamically tune protein structure and function in normal physiology and disease. With its abilities to serve as a surrogate for phosphorylation and other mechanisms of charge neutralization, and to potentially isolate early intermediates in protein assembly, this method may be useful for analysing never-before-seen early intermediates in the phosphorylation-driven assembly of other proteins in normal physiology and disease.

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

“…In the case of more complex biomolecules such as proteins, it might not be possible to probe all the protonatable amino acids in a single measurement. The method presented here is sensitive to amino acids that directly exchange charge with the bare electrode surface, where this electrochemical communication will depend on the tunneling barrier formed between the reacting amino acids and the electrode. − Evidence for the possibility to exchange charge between histidine residues inside a reflectin protein and a Pt surface was recently reported, emphasizing the larger scope and applicability of DPV as a valuable tool to probe amino acids’ proton dissociation equilibria in peptides and proteins.…”

“…Lastly, because different amino acid residues can be deprotonated under different applied potentials, the method presented enables identification of electrochemically accessible amino acids that can directly exchange charge with a bare Pt electrode surface. This, in turn, may allow one to electrochemically regulate the charge state of specific molecular moieties in proteins and peptides, beyond the results previously demonstrated for reflectin and polylysine, potentially allowing heterogeneous electrochemical control of Coulombic interactions often governing proteins’ structure.…”

“…Previous work has shown that platinum can catalyze electrochemical reduction of dissociable protons in a variety of molecules, ,, where the reduction potential is correlated with the p K a value of the proton-hosting chemical moiety via , …”

“…This correlation should be applicable to dissociable protons in freely diffusing charged amino acids, and potentially extendable to charged amino acid residues in peptides and proteins. If indeed the latter is true, electrochemistry would allow one to probe local chemical environments in peptides or proteins, as well as provide ways to actively trigger protonation-related, dynamic processes in biological molecules . It is worth noting that the equation above describes the equilibrium potential measured under standard conditions (25 °C, 1 atm, 1 M electrolyte) and is restricted to the limitations of the Nernst equation (activity coefficients of species are equal to 1 and no current is flowing through the system) and the Henderson–Hasselbalch equation (concentration of acid and its conjugate remains constant at equilibrium).…”

“…We recently demonstrated that low voltage applied to a platinum electrode can electrochemically reduce the imidazolium side chain of histidine when the amino acid is in the free state in solution, in oligopeptides, and in reflectin, a protein that transduces neuronal signals to regulate a biophotonic response. 1 In the case of reflectin, this electroreduction drives the condensation, secondary folding, and resulting hierarchical assembly that governs its biological activity in response to phosphorylation-mediated charge-neutralization. 1,2 This observation illustrates the fact that the dynamic control of conformation, assembly, and function of peptides and proteins is often governed by the charge states of their constituent amino acid residues.…”