Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

Abstract

Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site.

Protein bioelectronics: a review of what we do and do not know

“…318 With a similar approach, Bostick and co-workers used an indexed array of gold nanopillars as individual pixels for protein immobilization to which CYP2C9 could be linked using a SAM comprised of a mixed thiol layer. 145,146 Indexing of the array allowed multiple studies on the same P450 enzyme exposed to different substrates.…”

“…365 Force-dependent ETp investigations of Az, bR, CYP2C9, and other proteins after immobilizing onto gold substrates, have demonstrated added protein stability in the presence of cofactors or small molecule binders. 146 The response to an applied force of the conductance of an Az monolayer under cyclic mechanical loading (with CP-AFM) resembled memristive behavior, which could be modeled empirically using the viscoelastic property of the protein. Importantly, the Az conductance was found to depend not only on the force magnitude, but also on loading time.…”

Computational Studies on Structural, Excitation, and Charge-Transfer Properties of Ureidopeptidomimetics

“…This is due to the complexity in the structures of the bioentities and the ultrafast time scale in which charge is transfered. 25,26 Hence, biomimetics, such as modified peptides, 27 polypeptides, 28 and peptide nucleic acids, 29 have been synthesized and the CT mechanism explored, which provokes its comparison with signal transduction through a protein chain in biological systems. 30 Charge is transferred from the donor end to the acceptor end through a bridge, which is a covalent linkage in this case.…”

Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism

“…5). Immobilization of CYP2C9 controls its aggregation, with the majority being monomeric Bostick et al, 2015). Therefore, the inhibition of CYP2C9 metabolism could be a result of the lack of competition for CYP2C9 homomeric complex (CYP2C9-CYP2C9) formation with heteromeric complex (CYP2C9-CYP2D6) formation, evidenced by the similar K D values for both interactions (Table 3).…”

“…Controlled immobilization of P450s has recently been used to allow better understanding of metabolism (Davydov et al, 2005;Gannett et al, 2006;Mak et al, 2010;Fantuzzi et al, 2011;Panicco et al, 2011;Wollenberg et al, 2012;Bostick et al, 2015) and the formation of homomeric/heteromeric complexes (Backes and Kelley, 2003). We developed a scheme whereby soluble P450 is covalently attached to a self-assembled monolayer (SAM) on a gold film.…”