Characterization of the Membrane-Bound Full-Length Complex between Cytochrome b5 and Cytochrome P450 2B4.

Redox Complexes;Cytochrome B5;Cytochrome P450;Dynamic Complexes;NMR;HADDOCK;Biological Chemistry;Chemistry;Science;Chemistry

Le Clair, Stephanie ValerieMorris, Michael D. ;

University of Michigan

关键词: Redox Complexes;    Cytochrome B5;    Cytochrome P450;    Dynamic Complexes;    NMR;    HADDOCK;    Biological Chemistry;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/97880/leclairs_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

While cyt b5 has been shown to be an essential part of the catalytic cycle of cyt P450 in vivo, very little is known about its membrane-bound full-length structure and its ability to form complexes with cyt P450 in the membrane. Using Nuclear Magnetic Resonance (NMR) techniques, this dissertation describes the first characterization of full-length cyt b5 and its interaction with cyt P450 2B4 in a membrane environment. We present the structures of the soluble domain of cyt b5 and of the α-helical transmembrane domain, and identify the linker as being unstructured. Utilizing 15N relaxation measurements, the N-terminus and linker are shown to be highly flexible and additional internal dynamics are described. We then present a thorough investigation of the cyt b5-cyt P450 complex in isotropic bicelles. Cyt b5 interfacial residues are identified via NMR and mutagenesis, and a model of the cyt b5-cyt P450 complex is presented. From this model, non-covalent interactions at the interface are detailed, and we predict the electron pathway between the two proteins. A new solution NMR approach is then applied to the protein complex to further characterize the cyt b5-cyt P450 complex. We are able to propose a detailed mode of interaction between cyt b5 and cyt P450, identifying residues on the lower cleft of cyt b5 as being important for stereospecific complex formation and describing the extent of cyt b5 residues involved in encounter complexes. The addition of a cyt P450 substrate increases both the cyt b5 surface area scanned by cyt P450 and the population of encounter complexes. Using a mutant of cyt b5, in which the linker is truncated by eight residues (m-cyt b5), we show that the unstructured and dynamic linker region of wild-type cyt b5 allows cyt b5 to be able to sample more encounter complex orientations, essentially lengthening the lifetime of the encounter complex and increasing the probability of the two proteins finding a productive orientation (for electron transfer) within each macrocollision. This work yields new insights on the mechanisms of interaction between membrane-bound redox partners and the role of linker regions in these protein-protein interactions.