title: Proton-detected solid-state NMR reveals intramembrane polar networks in a seven-helical transmembrane protein proteorhodopsin.
abstract: We used high-resolution proton-detected multidimensional NMR to study the solvent-exposed parts of a seven-helical integral membrane proton pump, proteorhodopsin (PR). PR samples were prepared by growing the apoprotein on fully deuterated medium and reintroducing protons to solvent-accessible sites through exchange with protonated buffer. This preparation leads to NMR spectra with proton resolution down to ca. 0.2 ppm at fast spinning (28 kHz) in a protein back-exchanged at a level of 40%. Novel three-dimensional proton-detected chemical shift correlation spectroscopy allowed for the identification and resonance assignment of the solvent-exposed parts of the protein. Most of the observed residues are located at the membrane interface, but there are notable exceptions, particularly in helix G, where most of the residues are susceptible to H/D exchange. This helix contains Schiff base-forming Lys231, and many conserved polar residues in the extracellular half, such as Asn220, Tyr223, Asn224, Asp227, and Asn230. We proposed earlier that high mobility of the F-G loop may transiently expose a hydrophilic cavity in the extracellular half of the protein, similar to the one found in xanthorhodopsin. Solvent accessibility of helix G is in line with this hypothesis, implying that such a cavity may be a part of the proton-conducting pathway lined by this helix.
authors.affiliation: Department of Physics and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Meaghan E
authors.initials: ME
authors.lastname: Ward
authors.forename: Lichi
authors.initials: L
authors.lastname: Shi
authors.forename: Evelyn
authors.initials: E
authors.lastname: Lake
authors.forename: Sridevi
authors.initials: S
authors.lastname: Krishnamurthy
authors.forename: Howard
authors.initials: H
authors.lastname: Hutchins
authors.forename: Leonid S
authors.initials: LS
authors.lastname: Brown
authors.forename: Vladimir
authors.initials: V
authors.lastname: Ladizhansky
mesh_terms: D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000302:isolation & purification; D025604:Rhodopsins, Microbial
chemical_list: D011522:Protons; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin