Temperature-Dependent Solid-State NMR Proton Chemical-Shift Values and Hydrogen Bonding

Abstract

Temperature-dependent NMR experiments are often complicated by rather long magnetic-field equilibration times, for example, occurring upon a change of sample temperature. We demonstrate that the fast temporal stabilization of a magnetic field can be achieved by actively stabilizing the temperature of the magnet bore, which allows quantification of the weak temperature dependence of a proton chemical shift, which can be diagnostic for the presence of hydrogen bonds. Hydrogen bonding plays a central role in molecular recognition events from both fields, chemistry and biology. Their direct detection by standard structure-determination techniques, such as X-ray crystallography or cryo-electron microscopy, remains challenging due to the difficulties of approaching the required resolution, on the order of 1 Å. We, herein, explore a spectroscopic approach using solid-state NMR to identify protons engaged in hydrogen bonds and explore the measurement of proton chemical-shift temperature coefficients. Using the examples of a phosphorylated amino acid and the protein ubiquitin, we show that fast magic-angle spinning (MAS) experiments at 100 kHz yield sufficient resolution in proton-detected spectra to quantify the rather small chemical-shift changes upon temperature variations.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase

“…Due to their characteristic chemical shifts (and thus their isolated position in the 2D fingerprint spectrum) the temperature dependences could be directly extracted from 2D CP hNH experiments. It is well known from solution-state NMR that the chemical shifts of protons in strong intramolecular hydrogen bonds experience only a weak temperature dependence 60 , 61 as recently also shown by solid-state NMR 62 . However, for protons in rather weak hydrogen bonds, the resonances become significantly more shielded upon increasing the temperature, due to an increase in the average hydrogen-bond length.…”

“…A fast adjustment of the temperature in the bore of the magnet (typically causing B 0 instabilities) was achieved by a bore heating system implemented by the instrument manufacturer. This is crucial for detecting the rather small temperature dependences of proton chemical-shift values (on the order of several ppb/K) 62 . For 19 F (recorded at 14.1 T) and 27 Al (recorded at 11.7 T) MAS-NMR experiments, the sample temperature was adjusted to 278 K. 1 H and 31 P-detected spectra were analysed with the software CcpNmr (version 2.4.2) 94 – 96 and referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS).…”

“…We herein identify protein residues engaged in hydrogen bonding to the phosphate groups of nucleotides (ADP:AlF 4 − and DNA) by (i) measuring high-frequency shifted proton resonances characteristic for hydrogen-bond formation 58 , (ii) probing spatial proximities in dipolar-coupling based proton-detected 31 P, 1 H correlation experiments at fast MAS (105 kHz) and (iii) using the temperature dependence of 1 H chemical-shift values as a probe for hydrogen bonding, an approach well known in solution-state NMR 59 – 61 , and recently extended to the solid state 62 . Note, that we herein report a proton-detected 31 P, 1 H correlation spectrum at fast MAS frequencies using a sub-milligram sample amount, which was so far, to the best of our knowledge, not possible with any of the previous equipment.…”

Correction of field instabilities in biomolecular solid-state NMR by simultaneous acquisition of a frequency reference

“…Spectrometers typically provide a hardware linear drift correction that compensates for this field drift, which can be adjusted every few weeks or months by a calibration experiment. More importantly, insertion and removal of the NMR probe from the magnet bore and any changes in the temperature of the sample or the shim cylinder create a relatively strong non-linear drift that lasts, for high-field magnets, for hours (Malär et al, 2021), delaying the start of an experiment.…”

“…Alternatives to the field frequency lock approaches, that are applicable to solid-state NMR, too, have been used for reducing the field drift. Malär et al (2021) describe a spectrometer equipped with a magnet-bore heater system that prevents major long-term field drifts due to temperature changes inside the magnet bore, which greatly increases stability and shortens time constants to reach a stable field after a disturbance. External locks can monitor the 2 D or 7 Li resonance frequency of an auxiliary sample located in the proximity of the main sample, which is then utilized for the lock (Paulson and Zilm, 2005;Takahashi et al, 2012).…”