Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes

Abstract

Cytochrome P450 reductase (CPR) is a NADPH-dependent membranebound oxidoreductase found in the endoplasmic reticulum (ER) and is the main redox partner for most cytochrome P450 enzymes. Presented are the measured thermodynamic driving forces responsible for how strongly CPR partitions into a biomimetic ER with the same lipid composition of a natural ER. Using temperature-dependent fluorescence correlation spectroscopy and fluorescence single-protein tracking, the standard state free energies, enthalpies, and entropies of the CPR insertion process were all measured. The results of this study demonstrate that the thermodynamic driving forces are dependent on the redox states of CPR. In particular, the partitioning of CPR ox into a biomimetic ER is an exothermic process with a small positive change in entropy, while CPR red partitioning is endothermic with a large positive change in entropy. Both resulted in negative free energies and strong association to the biomimetic ER, but the K P of CPR ox insertion is measurably smaller than that of CPR red . Using this new information and known results from literature sources, we also present a phenomenological model that accounts for membrane−protein interactions, protein orientation relative to the membrane, and protein conformation as a function of the redox state.

Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes

“…Liposomes were composed of 98.6% 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine (POPC) and 1.4% 1,2-stearoyl- sn -glycerol-3-phosphoethanolamine-N-[methoxy­(polyethylene glycol)-2000] (ammonium salt) (PEG–PE). Liposomes were prepared as previously described. − Briefly, liposomes were prepared from lipid cakes made by evaporating 1 mL of a 9:1 chloroform: methanol solution that contained the above-outlined lipid mixtures. The solution was then evaporated overnight under a stream of prepurified nitrogen until no chloroform or methanol remained, and the lipid cake was thoroughly dry.…”

“…The exposure time for the camera was set to 50 ms and the frame rate was only slightly higher at 50.02 ms. Single-protein tracking analysis was done in MATLAB (MathWorks) using code based on work by Crocker and Grier using scripts modified and previously used by the authors. ,,, The vast majority of tracks were greater than 4 frames, with the average track lasting ∼24 frames (∼1.2 s) before photobleaching (recall that there are, on average, 4 fluorophores for each homotetramer). The few tracks that lasted four frames or less were considered too transient to be a transmembrane protein and likely caused by a fluorescent contaminate that reversibly interacted with the membrane.…”