Biophysics Program, Department of Chemistry, Macromolecular Science and Engineering, Biomedical Engineering, Michigan Neuroscience Institute, The University of Michigan, Ann Arbor, Michigan 48109-1055, United States.
Department of Pharmacology and Internal Medicine, Division of Metabolism, Endocrinology, & Diabetes, University of Michigan, Ann Arbor, Michigan 48109, United States.
Anal Chem. 2022 Aug 30;94(34):11908-11915. doi: 10.1021/acs.analchem.2c02489. Epub 2022 Aug 17.
Although polymer-based lipid nanodiscs are increasingly used in the structural studies of membrane proteins, the charge of the belt-forming polymer is a major limitation for functional reconstitution of membrane proteins possessing an opposite net charge to that of the polymer. This limitation also rules out the reconstitution of a protein-protein complex composed of oppositely charged membrane proteins. In this study, we report the first successful functional reconstitution of a membrane-bound redox complex constituting a cationic cytochrome P450 (CYP450) and an anionic cytochrome P450 reductase (CPR) in non-ionic inulin-based lipid nanodiscs. The gel-to-liquid-crystalline phase-transition temperature () of DMPC:DMPG (7:3 w/w) lipids in polymer nanodiscs was determined by differential scanning calorimetry (DSC) and P NMR experiments. The CYP450-CPR redox complex reconstitution in polymer nanodiscs was characterized by size-exclusion chromatography (SEC), and the electron transfer kinetics was carried out using the stopped-flow technique under anaerobic conditions. The of DMPC:DMPG (7:3 w/w) in polymer nanodiscs measured from P NMR agrees with that obtained from DSC and was found to be higher than that for liposomes due to the decreased cooperativity of lipids present in the nanodiscs. The stopped-flow measurements revealed the CYP450-CPR redox complex reconstituted in nanodiscs to be functional, and the electron transfer kinetics was found to be temperature-dependent. Based on the successful demonstration of the use of non-ionic inulin-based polymer nanodiscs reported in this study, we expect them to be useful in studying the function and structures of a variety of membrane proteins/complexes irrespective of the charge of the molecular components. Since the polymer nanodiscs were shown to align in an externally applied magnetic field, they can also be used to measure residual dipolar couplings (RDCs) and residual quadrupolar couplings (RQCs) for various molecules ranging from small molecules to soluble proteins and nucleic acids.
虽然基于聚合物的脂质纳米盘越来越多地用于膜蛋白的结构研究，但带形成聚合物的电荷是对与聚合物带相反电荷的膜蛋白进行功能重建的主要限制。这一限制也排除了由带相反电荷的膜蛋白组成的蛋白质-蛋白质复合物的重建。在这项研究中，我们报告了第一个成功的功能重建，即将阳离子细胞色素 P450（CYP450）和阴离子细胞色素 P450 还原酶（CPR）组成的膜结合氧化还原复合物重建到非离子型菊粉基脂质纳米盘中。通过差示扫描量热法（DSC）和 P NMR 实验测定了聚合物纳米盘中 DMPC：DMPG（7:3 w/w）脂质的凝胶到液晶相转变温度（T m）。通过凝胶过滤色谱（SEC）对聚合物纳米盘中 CYP450-CPR 氧化还原复合物的重建进行了表征，并在厌氧条件下使用停流技术进行了电子转移动力学研究。从 P NMR 测量的聚合物纳米盘中 DMPC：DMPG（7:3 w/w）的 T m与 DSC 获得的结果一致，并且由于纳米盘中存在的脂质的协同性降低，发现 T m高于脂质体。停流测量表明，在纳米盘中重建的 CYP450-CPR 氧化还原复合物具有功能，并且电子转移动力学是温度依赖性的。基于本研究中成功展示的使用非离子型菊粉基聚合物纳米盘，我们预计它们将有助于研究各种膜蛋白/复合物的功能和结构，而与分子成分的电荷无关。由于聚合物纳米盘在外加磁场中显示出排列，因此它们还可用于测量从小分子到可溶性蛋白质和核酸的各种分子的剩余偶极耦合（RDC）和剩余四极耦合（RQC）。
