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Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy

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  • Yuichi Yoshimura
  • ,
  • Nur Alia Oktaviani, University of Groningen
  • ,
  • Kento Yonezawa, Nara Institute of Science and Technology
  • ,
  • Hironari Kamikubo, Nara Institute of Science and Technology
  • ,
  • Frans A A Mulder

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.

Original languageEnglish
JournalAngewandte Chemie International Edition
Pages (from-to)239–242
Number of pages4
Publication statusPublished - 2 Jan 2017

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