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

TY - JOUR

T1 - Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy

AU - Yoshimura, Yuichi

AU - Oktaviani, Nur Alia

AU - Yonezawa, Kento

AU - Kamikubo, Hironari

AU - Mulder, Frans A A

N1 - © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

PY - 2017/1/2

Y1 - 2017/1/2

N2 - 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.

AB - 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.

U2 - 10.1002/anie.201609605

DO - 10.1002/anie.201609605

M3 - Journal article

C2 - 27897362

SN - 1433-7851

VL - 56

SP - 239

EP - 242

JO - Angewandte Chemie International Edition

JF - Angewandte Chemie International Edition

IS - 1

ER -