Premium
Reconstitution of the Cyt b 5 –CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy
Author(s) -
Zhang Meng,
Huang Rui,
Ackermann Rose,
Im SangChoul,
Waskell Lucy,
Schwendeman Anna,
Ramamoorthy Ayyalusamy
Publication year - 2016
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201600073
Subject(s) - nanodisc , nuclear magnetic resonance spectroscopy , chemistry , structural biology , cytochrome , peptide , membrane biology , two dimensional nuclear magnetic resonance spectroscopy , enzyme , membrane protein , membrane , biochemistry , stereochemistry
Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full‐length membrane‐bound P450 and its redox partner cytochrome b 5 (cyt b 5 ) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane‐bound complex in model membranes renders high‐resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450–cyt b 5 complex in peptide‐based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450–cyt b 5 complex in the nanodisc. This is the first successful demonstration of a protein–protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane‐bound protein complexes.