Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c

• Published in: Scientific reports 2017-08, Vol.7 (1)
• Main Authors: Gentry, Katherine A., Prade, Elke, Barnaba, Carlo, Zhang, Meng, Mahajan, Mukesh, Im, Sang-Choul, Anantharamaiah, G. M., Nagao, Satoshi, Waskell, Lucy, Ramamoorthy, Ayyalusamy
• Format: Article
• Language: English
• Subjects: 101/6; 140/131; 631/57/2271; 639/638/45/535/878/1263; 82; Humanities and Social Sciences; multidisciplinary; Science; Science (multidisciplinary)
• ISSN: 2045-2322
• DOI: 10.1038/s41598-017-08130-7

Cytochrome b 5 (cyt b 5 ) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cyt b 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cyt b 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cyt b 5 -cyt c binding interface. Our experimental results identify differences in the binding epitope of cyt b 5 in the presence and absence of membrane. Notably, in the presence of membrane, cyt b 5 only engaged cyt c at its lower and upper clefts while the membrane-free cyt b 5 also uses a distal region. Using restraints generated from both cyt b 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.