Determination of Histidine Protonation States in Proteins by Fast Magic Angle Spinning NMR.

Roman Zadorozhnyi,Caitlin M Quinn,Tatyana Polenova,Sucharita Sarkar,Kaneil K Zadrozny,Barbie K Ganser-Pornillos,Owen Pornillos,Angela M Gronenborn

doi:10.3389/fmolb.2021.767040

Abstract

Histidine residues play important structural and functional roles in proteins, such as serving as metal-binding ligands, mediating enzyme catalysis, and modulating proton channel activity. Many of these activities are modulated by the ionization state of the imidazole ring. Here we present a fast MAS NMR approach for the determination of protonation and tautomeric states of His at frequencies of 40–62 kHz. The experiments combine 1H detection with selective magnetization inversion techniques and transferred echo double resonance (TEDOR)–based filters, in 2D heteronuclear correlation experiments. We illustrate this approach using microcrystalline assemblies of HIV-1 CACTD-SP1 protein.

Highlights

Similar to solution NMR, the tautomeric state of histidines can be unambiguously determined from a unique combination of 15N sidechain chemical shifts
We report on a 2D 1H-detected transferred echo double resonance (TEDOR)-based Z-filtered experiment, which incorporates 15N selective filters for the determination of histidine tautomeric states
The tautomeric states of His residues are unambiguously determined using a combination of three CH HETCOR experiments comprising: i) 15N selective TEDOR filter, containing 13C resonances of all protonation and tautomeric states present; ii) 15N selective TEDOR filter with a soft pulse at 170 ppm, removing resonances of the protonated state while Cε1 and Cδ2 atoms of π tautomer and Cε1 and Cγ atoms of τ tautomer

Summary

Introduction

Histidines (His) play important structural and functional roles in proteins such as metal binding (Stryer et al, 1964; Perutz and Mathews, 1966; Adams et al, 1969; Liljas et al, 1972), proton transfer (Hoffee et al, 1967; Blow et al, 1969; Campbell et al, 1974), and stability (Perutz et al, 1969; Lewis et al, 1976; Loewenthal et al, 1992). For a protein at intermediate pH values, it is possible that a fraction of His residues is protonated and the remaining fraction unprotonated (French and Hammes, 1965; Edwards and Sykes, 1980; Hass et al, 2008)

Methods

Results

Conclusion