title: Solution NMR spectroscopy of [alpha -15N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence.
abstract: [alpha-(15)N]Lysine-labeled rhodopsin, prepared by expression of a synthetic gene in HEK293 cells, was investigated both by conventional and transverse relaxation optimized spectroscopy-type heteronuclear single quantum correlation spectroscopy. Whereas rhodopsin contains 11 lysines, 8 in cytoplasmic loops and 1 each in the C-terminal peptide sequence and the intradiscal and transmembrane domains, only a single sharp peak was observed in dodecyl maltoside micelles. This result did not change when dodecyl maltoside was replaced by octyl glucoside or octyl glucoside-phospholipid-mixed micelles. Additional signals of much lower and variable intensity appeared at temperatures above 20 degrees C and under denaturing conditions. Application of the transverse relaxation optimized spectroscopy sequence resulted in sharpening of resonances but also losses of signal intensity. The single peak observed has been assigned to the C-terminal Lys-339 from the following lines of evidence. First, the signal is observed in HNCO spectra of rhodopsin, containing the labeled [(13)C]Ser-338/[(15)N]Lys-339 dipeptide. Second, addition of a monoclonal anti-rhodopsin antibody that binds to the C-terminal 8 aa of rhodopsin caused disappearance of the peak. Third, truncated rhodopsin lacking the C-terminal sequence Asp-330-Ala-348 showed no signal, whereas the enzymatically produced peptide fragment containing the above sequence showed the single peak. The results indicate motion in the backbone amide groups of rhodopsin at time scales depending on their location in the sequence. At the C terminus, conformational averaging occurs at the nanosecond time scale but varies from microsecond to millisecond in other parts of the primary sequence. The motions reflecting conformational exchange may be general for membrane proteins containing transmembrane helical bundles.
authors.affiliation: School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
authors.forename: J
authors.initials: J
authors.lastname: Klein-Seetharaman
authors.forename: P J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: M C
authors.initials: MC
authors.lastname: Loewen
authors.forename: E V
authors.initials: EV
authors.lastname: Getmanova
authors.forename: J
authors.initials: J
authors.lastname: Chung
authors.forename: H
authors.initials: H
authors.lastname: Schwalbe
authors.forename: P E
authors.initials: PE
authors.lastname: Wright
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D003902:Detergents; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D008027:Light; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D003902:Detergents; D004220:Disulfides; D008823:Micelles; D012996:Solutions; D012243:Rhodopsin; D008239:Lysine