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

Yuichi Yoshimura, Nur Alia Oktaviani, Kento Yonezawa, Hironari Kamikubo, Frans A. A. Mulder*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

23 Citations (Scopus)

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 C-13-detected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects. In the experiment, the N-15(eta) and N-15(epsilon) chemical shifts of an arginine head group are correlated with that of the directly attached C-13(zeta). In the resulting spectrum, the number of protons in the arginine head group can be obtained directly from the N-15-H-1 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-eta atoms were previously identified by neutron crystallography, we show that R52 is predominantly protonated in solution.

Original languageEnglish
Pages (from-to)239-242
Number of pages4
JournalAngewandte Chemie-International Edition
Volume56
Issue number1
DOIs
Publication statusPublished - 2-Jan-2017

Keywords

  • arginine
  • cross polarization
  • N-15-H-1 spin-spin coupling
  • NMR spectroscopy
  • photoactive yellow protein
  • PHOTOACTIVE YELLOW PROTEIN
  • INTRINSICALLY DISORDERED PROTEINS
  • SIDE-CHAIN DYNAMICS
  • HYDROGEN-BONDS
  • ACTIVE-SITE
  • N-15
  • RESIDUES
  • LYSINE
  • BACTERIORHODOPSIN
  • THERMOSTABILITY

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