Measuring radiofrequency fields in NMR spectroscopy using offset-dependent nutation profiles

Abstract

The application of NMR spectroscopy for studying molecular and reaction dynamics relies crucially on the measurement of the magnitude of radiofrequency (RF) fields that are used to nutate or lock the nuclear magnetization. Here, we report a method for measuring RF field amplitudes that leverages the intrinsic modulations observed in offset-dependent NMR nutation profiles of small molecules. Such nutation profiles are exquisitely sensitive to the magnitude of the RF field, and B 1 values ranging from 1 to 2000 Hz, as well the inhomogeneity in B 1 distributions, can be determined with high accuracy and precision using this approach. In order to measure B 1 fields associated with NMR experiments carried out on protein or nucleic acids, where these modulations are obscured by the large transverse relaxation rate constants of the analyte, our approach can be used in conjunction with a suitable external small molecule standard, expanding the scope of the method for large biomolecules.

Measuring Hydroxyl Exchange Rates in Glycans Using a Synergistic Combination of Saturation Transfer and Relaxation Dispersion NMR

“…We therefore adopted a selective 1D excitation scheme to interrogate peaks one at a time in the context of either a CEST or a CPMG experiment (Figure C). In this scheme, selective excitation is achieved through a Hartmann–Hahn J-cross-polarization module, which employs weak radiofrequency (RF) fields with matching amplitudes on both the 1 H and 13 C channels applied on-resonance to the respective 1 H and 13 C chemical shifts of a 1 H– 13 C covalently bonded spin pair. ,, When RF field strengths of the order of 1 J HC are used ( 1 J HC ∼ 145 Hz, B 1 ∼ 130 Hz for methine carbons and 1 J HC ∼ 125 Hz, B 1 ∼ 115 Hz for methylene carbons), magnetization is transferred from the hydrogen of interest to its covalently bonded 13 C nucleus, resulting in a single peak in the 13 C-edited selective 1D 1 H spectrum. ,, Selective 1D CEST and R 1ρ pulse sequences have previously been used for probing protein and nucleic acid dynamics, ,,, as well as to measure RF amplitudes Figure D shows the 1D 1 H spectrum of sucrose in red, overlaid with a selective 1D spectrum exciting either the FC3-H3 methine (Figure D, blue) or the GC6-H6 methylene (Figure D, green) spin systems.…”

“…It is therefore important to acquire data at B 1 fields ≤5 Hz in order to reliably determine the parameters quantifying the exchange event. Note that the two dips in intensity in the 5 Hz CEST profile of GC6 originate from offset-dependent nutation and not from H/D exchange (SI Text, Figure S5). Figure B shows 13 C-CPMG profiles acquired on the same sample at a static field strength of 16.45 T. Chemical exchange between the C–OH and C–OD states separated in chemical shift by 2 Δ 13 C­(D) results in CPMG dispersions ranging in magnitude (Δ R 2 eff = R 2 eff ( v CPMG = 25 Hz) – R 2 eff (ν CPMG,max )) from 3.5–5 s –1 .…”

“…It is therefore important to acquire data at B 1 fields ≤5 Hz in order to reliably determine the parameters quantifying the exchange event. Note that the two dips in intensity in the 5 Hz CEST profile of GC6 originate from offset-dependent nutation 32 and not from H/D exchange (SI Text, Figure S5). Figure 2B shows…”

“…24,25,28 Selective 1D CEST and R 1ρ pulse sequences have previously been used for probing protein and nucleic acid dynamics, 25,28,30,31 as well as to measure RF amplitudes. 32 Figure 1D shows the 1D 1 H spectrum of sucrose in red, overlaid with a selective 1D spectrum exciting either the FC3-H3 methine (Figure 1D, blue) or the GC6-H6 methylene (Figure 1D, green) spin systems. A conventional 2D-based CEST experiment on sucrose would take 184 h for acquiring 736 planes (92 planes centered around each of the eight nonoverlapping carbons attached to hydroxyls) spaced 2 Hz apart (4 transients per increment for each offset) covering the entire 13 C sweep width.…”

“…39 Therefore, artifactual contributions of crossrelaxation to the exchange rate constant measurements are negligible. Finally, owing to the fact that small B 1 fields are employed in the 13 C-CEST experiment, B 1 fields were calibrated using the CONDENZ method, 32 as this allows for precise and accurate measurements of small B 1 field strengths.…”