Bioelectrocatalytic Electrodes Modified with PQQ‐Glucose Dehydrogenase‐Calmodulin Chimera Switchable by Peptide Signals: Pathway to Generic Bioelectronic Systems Controlled by Biomolecular Inputs

Abstract

Construction of artificial allosteric protein switches is one of the central goals of synthetic biology that holds promise to transform the way we detect and quantify substances in vitro and in vivo. An artificial chimeric fusion protein of pyrroloquinoline quinone‐dependent glucose dehydrogenase with calmodulin (PQQ‐GDH‐CaM) was covalently attached to graphene nanosheets produced electrochemically on a carbon fiber electrode. The chimeric PQQ‐GDH‐CaM represents an artificial allosteric switch activated by association of a calmodulin‐binding peptide with the Ca2+‐bound calmodulin domain. The activity of the immobilized enzyme was switched between active and inactive states by adding/removing the activating peptide. The peptide‐signal switchable features originated from the enzyme 3D‐structural variations induced by the conformational (folding/unfolding) changes in the connected calmodulin unit upon formation/dissociation of its complex with the specific peptide. The peptide‐activated immobilized PQQ‐GDH‐CaM enzyme displayed direct (non‐mediated) electron transfer to the conducting electrode support upon glucose oxidation. On the contrary, in the absence of the peptide, the inactive form of the enzyme demonstrated very low bioelectrocatalytic activity for glucose oxidation. Since the conformational changes of the PQQ‐GDH‐CaM depend on the presence of Ca2+ cations and the calmodulin‐binding peptide, both of them were used as input signals to control the enzyme activity mimicking a Boolean AND logic gate. The switchable behavior of the enzyme‐modified electrode was studied electrochemically and used to assemble a signal‐switchable biofuel cell. The use of the peptide as the signaling messenger enables the design of generalizable bioelectronic systems controlled by native and synthetic biochemical signaling systems.

Connecting Artificial Proteolytic and Electrochemical Signaling Systems with Caged Messenger Peptides

“…Electrochemical analysis of the electrode-immobilized system reveals that it is catalytically inactive and does not produce current when glucose is present but the CaM-BP is absent (Figure A). , Figure A shows a set of cyclic voltammograms (CVs) obtained upon the stepwise addition of the proteolytic biosensor components required for the release of the CaM-BP and subsequent activation of the GDH-CaM-modified electrode. As expected, the system remained electrocatalytically inert (Figure A, curves a–e) until all components of the system were added, allowing rapamycin to trigger CaM-BP release from the calmodulin cage.…”

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals

“…The CaM receptor unit changes its conformation dramatically upon binding Ca 2+ cations and M13 peptide cooperatively, Figure A. This conformation change transfers to the PQQ‐GDH enzyme backbone affecting the biocatalytic activity . The PQQ‐GDH‐CaM chimeric enzyme is catalytically inactive when the CaM receptor unit exists in the extended state in the absence of M13 peptide, Figure A.…”

“…This conformation change transfers to the PQQ-GDH enzyme backbone affecting the biocatalytic activity. [31,32] The PQQ-GDH-CaM chimeric enzyme is catalytically inactive when the CaM receptor unit exists in the extended state in the absence of M13 peptide, Figure 2A. On the other hand, when the CaM receptor 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 binds the M13 peptide and changes its conformation to the folded state, Figure 1A, the PQQ-GDH enzyme conformation is also changed and the enzyme is activated for glucose oxidation, Figure 2A.…”

A magneto-controlled biocatalytic cascade with a fluorescent output

“…The signal-recognition units attached to enzymes occur in the form of photo-isomerizable species (e.g., spiropyran ↔ merocyanine) responding to light 4,5 or biomolecules isomerized upon complexation with molecular signals (e.g., calmodulin). 6,7 These units were integrated with enzymes simply through their covalent binding or through complex genetic engineering resulting in chimeric enzymes. The conformational changes in the bound signal-recognition units are transduced to the enzyme molecules, which change their activity.…”