Expression, purification, and functional reconstitution of 19F-labeled cytochrome b5 in peptide nanodiscs for NMR studies

Academic Article

Abstract

  • Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of 19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. The reported results demonstrate the use of 19F NMR experiments to study 19F-labeled (with 5-fluorotryptophan (5FW)) cytb5 reconstituted in peptide-nanodiscs and the detection of residues from the transmembrane domain by solution 19F NMR experiments. 19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular 19F-19F distance constraints to determine the structural interactions between the transmembrane domains.
  • Digital Object Identifier (doi)

    Author List

  • Bai J; Wang J; Ravula T; Im SC; Anantharamaiah GM; Waskell L; Ramamoorthy A
  • Volume

  • 1862
  • Issue

  • 5