Membrane‐Mediated Protein–Protein Interactions of Cholesterol Side‐Chain Cleavage Cytochrome P450 with its Associated Electron Transport Proteins

Abstract

Cytochrome P450scc (P450scc or CYP11A1) catalyses the first enzymatic step of steroid biosynthesis, the cleavage of the side chain of cholesterol to produce pregnenolone in the mitochondrion. The activity of P450scc is dependent upon electron delivery from NADPH‐dependent adrenodoxin reductase (AdR), via adrenodoxin (Adx), to the P450scc. However, despite the structural and kinetic data that supports the mechanism by which Adx shuttles electrons one at a time between AdR and the P450scc, there are limited data available on the influence of the lipid membrane on these essential interactions. In this paper, the protein–membrane interactions between P450scc and its redox partners were examined on 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) membranes containing cholesterol (20 %), using a quartz crystal microbalance with dissipation monitoring. P450scc showed strong binding to these membranes, whereas AdR and Adx both showed weaker association. If pre‐mixed, all three proteins bound independently to the membrane layer in a distinctive two‐stage process, as observed by frequency changes upon binding. Concomitant changes in the dissipation revealed specific protein–protein interaction occurs upon reaching a critical concentration of proteins in the membrane layer. These changes were specific for the binding of the three pre‐mixed proteins and were not observed for a binary mixture of P450 and Adx, or sequential binding of the three proteins. A simple model was developed for the binding of all three proteins in a 1:1:1 mixture to the membrane and reproduces the experimental data describing the interaction of P450scc with the other proteins (AdR and Adx) after initial binding of the individual proteins. Thus, we conclude that the lipid membrane assists in the assembly of electron transport proteins and the activity of P450scc by providing a surface for the localised concentration of proteins, enabling them to act together as a metabolon.

Amyloid aggregation and membrane activity of the antimicrobial peptide uperin 3.5

“…Each QCM‐D experiment was carried out using the Q‐Sense E4 (Q‐Sense, Stockholm, Sweden) system. In brief, sensors were cleaned, liposomes prepared ex situ and deposited onto the sensors to form a lipid bilayer as described previously …”

“…The harmonics (third, fifth, seventh, and ninth) of the sensor's resonance frequency penetrate the lipid bilayer inversely to the overtone number, enabling a depth profile to be obtained. As a result, conclusions regarding the structural organization or mechanism of peptide binding to the membrane can be gleaned …”

Effects of guanidino modified aminoglycosides on mammalian membranes studied using a quartz crystal microbalance

“…The lipid bilayer formation is verified by a characteristic change in frequency (Δf = −26 ± 5 Hz) and dissipation (ΔD = (4 ± 1) × 10 -6 ) of the supported lipid bilayer. 38 Note that the change in dissipation is higher for SAM-modified gold sensors than for SiO 2 sensors. [38][39][40] After a supported lipid bilayer was formed, the aminoglycoside samples were continuously introduced at 50 μL min −1 for about one hour.…”

“…38 Note that the change in dissipation is higher for SAM-modified gold sensors than for SiO 2 sensors. [38][39][40] After a supported lipid bilayer was formed, the aminoglycoside samples were continuously introduced at 50 μL min −1 for about one hour. The system was finally rinsed with high salt PBS buffer.…”

“…[33][34][35][36][37] Mass changes due to drug binding to the surface cause frequency changes of the QCM sensor. [38][39][40][41] The technique is based upon the piezoelectric effect of single quartz crystals. The fundamental relationship between the added mass Δm and the change in resonance frequency Δf n was described for rigid, thin and evenly distributed adsorbed layers by Sauerbrey in 1959.…”