Molecular LEGO by domain-imprinting of cytochrome P450 BM3
| Autor: | Anna Fischer, Vlada B. Urlacher, Aysu Yarman, Inez M. Weidinger, Katharina J. Jetzschmann, Ulla Wollenberger, Lina Rustam, Patrycja Kielb, Frieder W. Scheller |
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| Rok vydání: | 2018 |
| Předmět: |
Polymers
Protein domain 02 engineering and technology 010402 general chemistry 01 natural sciences Fluorescence Molecular Imprinting chemistry.chemical_compound Colloid and Surface Chemistry Bacterial Proteins Cytochrome P-450 Enzyme System Protein Domains ddc:570 Animals Horses Physical and Theoretical Chemistry Ferricyanides Institut für Biochemie und Biologie NADPH-Ferrihemoprotein Reductase biology Cytochrome c Molecularly imprinted polymer Surfaces and Interfaces General Medicine 021001 nanoscience & nanotechnology 0104 chemical sciences Solutions Monomer chemistry Biophysics biology.protein NIP Ferricyanide Cyclic voltammetry 0210 nano-technology Molecular imprinting Biotechnology |
| Zdroj: | Colloids and Surfaces B: Biointerfaces. 164:240-246 |
| ISSN: | 0927-7765 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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