Structural Study of Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Its Redox Partners – Cytochrome P450/Cytochrome c by NMR and NMR Characterization of Monomeric and Oligomeric Conformations of Human Calcitonin and Its Interaction with EGCG.

[摘要] Cytochrome P450 reductase (CPR) is a diflavin protein which donates electrons to a variety of microsomal cytochrome P450s, heme oxygenase, cytochrome b5, cytochrome c as well as therapeutic prodrugs. It plays an indispensable role in the cytochrome P450 monooxygenase system, which is responsible for the metabolism of myriads of endogenous compounds as well as exogenous compounds. In order to understand the electron transfer mechanism between CPR and its redox partners and get an better insight into its physiological function, it is essential to obtain detailed structural information on the electron transfer complexes. This thesis aims to unveil the structures of electron transfer complexes formed between the FMN binding domain (FBD) of CPR and its redox partners – cytochrome P450 and cytochrome c, map the binding interfaces of the complexes and propose potential electron transfer pathways. A combination of solution and solid-state NMR techniques, in conjunction with kinetics studies and molecular docking, were utilized for the investigation. Kinetics of electron transfer from FBD to cytochrome P450 and cytochrome c was characterized and compared with wild-type CPR. The results support that conformational gating of CPR plays a key role in the kinetics of interprotein electron transfer. Solution NMR experiments were carried out to map the binding interfaces in the complexes, from which the structural models of the complexes were generated. In addition, high-resolution structures of human calcitonin have been investigated using NMR spectroscopy. Calcitonin is a peptide hormone known for its hypocalcemic effect and its inhibition of bone resorption. While calcitonin has been used in therapy for osteoporosis and Paget;;s disease for decades, human calcitonin (hCT) forms fibrils in aqueous solution that limit its therapeutic application. The molecular mechanism of fiber formation by calcitonin is not well understood.In this thesis, by solving the high-resolution structures of hCT under two different conditions, we discovered that the peptide undergoes a conformational transition in the process of molecular association. The effect of the polyphenol epigallocatechin 3-gallate (EGCG) on hCT fibrillation was also investigated, which show that EGCG efficiently inhibits fibril formation of hCT by preventing the initial association of hCT before fiber formation.

[发布机构] University of Michigan

[效力级别] Electron-transfer Protein Complex

[关键词] Cytochrome P450 Reductase;Electron-transfer Protein Complex;NMR;Human Calcitonin;Amyloidogenic Peptide;EGCG Amyloid Inhibitor;Chemistry;Science;Chemistry