title: Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy.
abstract: Because arginine residues in proteins are expected to be in their protonated form almost without exception, reports demonstrating that a protein arginine residue is charge-neutral are rare and potentially controversial. Herein, we present a13C-detected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects. In the experiment, the15Nηand15Nϵchemical shifts of an arginine head group are correlated with that of the directly attached13Cζ. In the resulting spectrum, the number of protons in the arginine head group can be obtained directly from the15N-1H scalar coupling splitting pattern. We applied this method to unambiguously determine the ionization state of the R52 side chain in the photoactive yellow protein from Halorhodospira halophila. Although only three Hηatoms were previously identified by neutron crystallography, we show that R52 is predominantly protonated in solution.
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark.
authors.forename: Yuichi
authors.identifier: 0000-0002-9905-453X
authors.initials: Y
authors.lastname: Yoshimura
authors.affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors.forename: Nur Alia
authors.identifier: 0000-0002-7948-5949
authors.initials: NA
authors.lastname: Oktaviani
authors.affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
authors.forename: Kento
authors.initials: K
authors.lastname: Yonezawa
authors.affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
authors.forename: Hironari
authors.identifier: 0000-0002-0878-6488
authors.initials: H
authors.lastname: Kamikubo
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark.|Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors.forename: Frans A A
authors.identifier: 0000-0001-9427-8406
authors.initials: FA
authors.lastname: Mulder
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D020607:Halorhodospira halophila / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D020283:Photoreceptors, Microbial / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria; D001120:Arginine
keywords: 15N-1H spin-spin coupling; NMR spectroscopy; arginine; cross polarization; photoactive yellow protein