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Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy
Author(s) -
Yoshimura Yuichi,
Oktaviani Nur Alia,
Yonezawa Kento,
Kamikubo Hironari,
Mulder Frans A. A.
Publication year - 2017
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201609605
Subject(s) - protonation , arginine , chemistry , nuclear magnetic resonance spectroscopy , crystallography , residue (chemistry) , spectroscopy , ionization , chemical shift , side chain , stereochemistry , amino acid , ion , organic chemistry , biochemistry , physics , polymer , quantum mechanics
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 a 13 C‐detected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects. In the experiment, the 15 N η and 15 N ϵ chemical shifts of an arginine head group are correlated with that of the directly attached 13 C ζ . In the resulting spectrum, the number of protons in the arginine head group can be obtained directly from the 15 N– 1 H 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 H η atoms were previously identified by neutron crystallography, we show that R52 is predominantly protonated in solution.