K J Jetzschmann1, A Yarman2, L Rustam3, P Kielb4, V B Urlacher5, A Fischer3, I M Weidinger6, U Wollenberger1, F W Scheller7. 1. Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany. 2. Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany; Faculty of Science, Molecular Biotechnology, Turkish-German University, Sahinkaya Cad. 86, 34820 Beykoz, Istanbul, Turkey. 3. Institut für Anorganische und Analytische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104 Freiburg, Germany. 4. Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany. 5. Institute of Biochemistry, Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany. 6. Department of Chemistry and Food Chemistry, Technische Universität Dresden, Zellescher Weg 19, 01069 Dresden, Germany. 7. Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany. Electronic address: fschell@uni-potsdam.de.
Abstract
HYPOTHESIS: Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. EXPERIMENTS: Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). FINDINGS: The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his6-tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his6-tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode.
HYPOTHESIS: Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. EXPERIMENTS: Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). FINDINGS: The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his6-tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his6-tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode.
Authors: Robert D Crapnell; Alexander Hudson; Christopher W Foster; Kasper Eersels; Bart van Grinsven; Thomas J Cleij; Craig E Banks; Marloes Peeters Journal: Sensors (Basel) Date: 2019-03-09 Impact factor: 3.576