The CYP 450 superfamily of enzymes is classified as peripheral membrane proteins and is surrounded by various lipids in their vicinity. While previous studies have underlined the importance of these lipids, the role and mechanism through which the lipid composition modulates the physiochemical properties of CYP 450s is a lesser explored question. In this study, CYP2J2 was reconstituted into nanodiscs of different lipid compositions using the lipids found in the endoplasmic reticulum (ER) membrane. Using a combination of nano-differential scanning fluorimetry and UV-visible spectroscopy, we demonstrate that CYP2J2 undergoes a transition in its unfolding behavior between the detergent micelle and the nanodisc environment, with the first melting transition corresponding to significant heme dissociation. Further, we show that changing the lipid environment produced a shift of up to 3–4°C and 8–9°C for the first and second melting transition temperatures, respectively, with sphingomyelin NDs and POPS (1-palmitoyl-2-oleoyl-glycero-3-phosphoserine) NDs showing maximum and minimum thermal stabilities, respectively. A functional assay using a novel fluorescent probe showed that lipid composition directly impacts the function of the protein, with POPS and sphingomyelin NDs showing maximum and minimum catalytic activity. Lipid composition was found to have minimal effects on ligand binding affinities. However, NADPH-oxidation rates showed that lipid composition directly affects the function by altering the rate of electron transfer between the CYP and its redox partner cytochrome P450 reductase (CPR). Fluorescence anisotropy measurements show that along with the charge of the lipid head-group, membrane fluidity is an important factor in our understanding of how lipid composition exerts its effects. Overall, this study aims to study CYP2J2 in nanodiscs of varying lipid composition and delineate the impact of protein-lipid interactions and membrane fluidity on the function and stability of membrane proteins using biophysical methods.
Das et al. (Sun,) studied this question.