Open AccessKinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
Author(s) -
Katherine A. Gentry,
Elke Prade,
Carlo Barnaba,
Meng Zhang,
Mukesh Mahajan,
Sang Choul Im,
G.M. Anantharamaiah,
Shigeaki Nagao,
Lucy Waskell,
Ayyalusamy Ramamoorthy
Publication year - 2017
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/s41598-017-08130-7
Subject(s) - cytochrome c , cytochrome , cytochrome b , electron transfer , chemistry , membrane protein , membrane , redox , bacterial outer membrane , biophysics , crystallography , biology , biochemistry , mitochondrion , enzyme , mitochondrial dna , photochemistry , gene , organic chemistry , escherichia coli
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.