Protein and Electrode Engineering for the Covalent Immobilization of P450 BMP on Gold

Abstract

Site-directed mutagenesis and functionalization of gold surfaces have been combined to obtain a stable immobilization of the heme domain of cytochrome P450 BM3 from Bacillus megaterium. Immobilization experiments were carried out using the wild type protein bearing the surface C62 and C156 and the site-directed mutants C62S, the C156S, and the double mutant C62S/C156S (no exposed cysteines). The gold surface was functionalized using two different spacers: cystamine- N-succinimidyl 3-maleimidopropionate and dithio-bismaleimidoethane, both leading to the formation of maleimide-terminated monolayers capable of covalent linkage to cysteine. Tapping mode atomic force microscopy experiments carried out on cystamine- N-succinimidyl 3-maleimidopropionate derivatized gold led to good images with expected molecular heights (5.5-6.0 nm) for the wild type and the C156S mutant. These samples also gave measurable electrochemical signals with midpoint potentials of -48 and -58 mV for the wild type and C156S, respectively. On the other hand, the dithio-bismaleimidoethane spacer led to variability on the molecular heights measured by tapping mode atomic force microscopy and the electrochemical response. This is interpreted in terms of lack of homogeneous dithio-bismaleimidoethane monolayer on gold. Furthermore, results from tapping mode atomic force microscopy show that the double mutant and the C62S did not lead to stably immobilized P450 protein, confirming the necessity of the solvent exposed C62.

Functionalizable low-fouling coatings for label-free biosensing in complex biological media: advances and applications

“…Thiol groups of cysteine residues located in the constant F ab domain of antibody fragments or at selected sites of recombinant proteins and polypeptides can be used for covalent binding of the bioreceptors to maleimide-functionalized surfaces without a decrease in their analyte-binding activity [66,67]. The immobilization of an antibody via covalent binding of an oxidized oligosaccharide located in the F c region of an antibody to hydrazine or hydroxylamine-functionalized surfaces enables oriented antibody immobilization without decreasing the antigen-binding activity of the F ab regions.…”

Bioelectronic Delivery of Electrons to Cytochrome P450 Enzymes

“…40 Covalent immobilization includes the use of self-assembled monolayers, 41 and electrodes modified with maleimide group to orient cyt P450s via cysteine residues. 42-45 Scheller reviewed direct electron transfer studies and electrocatalysis of cyt P450s up to 2004. 20 …”

Engineering Macaca fascicularis cytochrome P450 2C20 to reduce animal testing for new drugs

“…Gold electrodes (11 mm × 5 mm, custom made by Arrandee, Germany) consisting of a vacuum evaporated thin gold film (thickness 200 nm) on borosilicate glass were annealed with a butane flame to obtain flat re-crystallized Au(111) terraces and were functionalized with dithio-bismaleimidoethane (DTME) as reported previously [12,[21][22][23]. This functionalization led to the formation of maleimide-terminated groups that subsequently interacted with exposed cysteine residues present on the surface of the M. fascicularis P450 2C20 (30 μM) resulting in covalent linkage of the protein to the Au surface.…”