title: Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization.
abstract: While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can provide 3D structural information. However, there are numerous challenges to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges in order to obtain high-resolution structural insights into electron transfer processes mediated by membrane-bound proteins like mammalian cytochrome-b5, cytochrome-P450 and cytochrome-P450-reductase. In this study, we demonstrate the feasibility of using dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from ¹³C-labeled membrane-anchored cytochrome-b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement. Further, results obtained from a 2D ¹³C/¹³C chemical shift correlation MAS experiment demonstrate the feasibility of suppressing the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes.
authors.affiliation: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Bruker BioSpin Corporation, 15 Fortune Drive, Billerica, MA 01821, USA.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Caporini
authors.affiliation: Department of Anesthesiology, University of Michigan, Ann Arbor, MI 48105, USA; VA Medical Center, Ann Arbor, MI 48105, USA.
authors.forename: Sang-Choul
authors.initials: SC
authors.lastname: Im
authors.affiliation: Department of Anesthesiology, University of Michigan, Ann Arbor, MI 48105, USA; VA Medical Center, Ann Arbor, MI 48105, USA.
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
authors.affiliation: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA. Electronic address: ramamoor@umich.edu.
authors.forename: Ayyalusamy
authors.initials: A
authors.lastname: Ramamoorthy
mesh_terms: D000595:Amino Acid Sequence; D015786:Cytochromes b5 / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data
chemical_list: D008565:Membrane Proteins; D015786:Cytochromes b5
keywords: Dynamic nuclear polarization; In-cell NMR spectroscopy; Lipid bilayers; Membrane protein; Solid-state NMR spectroscopy; Structural biology