Molecular LEGO by domain-imprinting of cytochrome P450 BM3

• Published in: Colloids and surfaces, B, Biointerfaces B, Biointerfaces, 2018-04, Vol.164, p.240-246
• Main Authors: Jetzschmann, K.J., Yarman, A., Rustam, L., Kielb, P., Urlacher, V.B., Fischer, A., Weidinger, I.M., Wollenberger, U., Scheller, F.W.
• Format: Article
• Language: English
• Subjects: Cytochrome P450; Electropolymerization; Molecularly imprinted polymers; Protein imprinting
• ISSN: 0927-7765; 1873-4367
• DOI: 10.1016/j.colsurfb.2018.01.047

Simplified presentation of the LEGO-like MIP-interaction with the BM3-domains: The MIP synthesized with the BMR-domain recognizes its target whilst the BMO-domain and the holoenzyme are not bound. Obviously, a cavity is formed during the imprinting process which cannot accommodate the broader BMO. [Display omitted] 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. 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). 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.