TmDOTP: An NMR-based thermometer for magic angle spinning NMR experiments

Abstract

Solid state NMR is a powerful tool to probe membrane protein structure and dynamics in native lipid membranes. Sample heating during solid state NMR experiments can be caused by magic angle spinning and radio frequency irradiation such heating produces uncertainties in the sample temperature and temperature distribution, which can in turn lead to line broadening and sample deterioration. To measure sample temperatures in real time and to quantify thermal gradients and their dependence on radio frequency irradiation or spinning frequency, we use the chemical shift thermometer TmDOTP, a lanthanide complex. The H₆ TmDOTP proton NMR peak has a large chemical shift (-176.3 ppm at 275 K) and it is well resolved from the protein and lipid proton spectrum. Compared to other NMR thermometers (e.g., the proton NMR signal of water), the proton spectrum of TmDOTP, particularly the H₆ proton line, exhibits very high thermal sensitivity and resolution. In MAS studies of proteoliposomes we identify two populations of TmDOTP with differing temperatures and dependency on the radio frequency irradiation power. We interpret these populations as arising from the supernatant and the pellet, which is sedimented during sample spinning. In this study, we demonstrate that TmDOTP is an excellent internal standard for monitoring real-time temperatures of biopolymers without changing their properties or obscuring their spectra. Real time temperature calibration is expected to be important for the interpretation of dynamics and other properties of biopolymers.

Keywords: Dielectric loss; Magic-angle spinning; Nuclear magnetic resonance; Real-time NMR temperature measurement; Sample Heating; TmDOTP(5-).

Fig. 1.

(A) Molecular structure of TmDOTP with H₆ highlighted and portion of the ¹H NMR spectrum of 25 mM TmDOTP. The sample contains KcsA proteoliposome and 25 mM TmDOTP. H₆ is at −176.3 ppm while H₁ is −218.8 ppm. (B) Overlay of the spectra of the H₆ proton in TmDOTP acquired at various temperatures. All spectra were collected on 900 MHz with MAS frequency at 5 kHz. Chemical shift was referenced to the DSS at 0 ppm.

Fig. 2.

¹H pulse sequence used to measure the temperature increase from RF irradiation. The RF irradiation is applied for the duration of τ₁. τ₂ represents the delay before proton 90 pulse. A spin echo with τ₃ = 20 μs is applied before acquisition to dephase the signal from H₁ in TmDOTP.

Fig. 3.

The temperature dependence of the chemical shift of the H₆ proton in TmDOTP (blue) and of the water proton (orange). Δδ_iso is the change in chemical shift relative to the shift at VT gas temperature of 275 K. All spectra were collected on a Bruker Avance II 900 MHz spectrometer equipped with a 3.2 mm standard-bore E-free probe. The spinning frequency was 5 kHz and the gas flow rate was 1070 L/h. The dashed lines represent a linear least squares optimized fit to the data: ${\delta \mathit{iso}}_{,\mathit{TmDOTP}}=1.06(\frac{\mathit{ppm}}{K})T-291(\mathrm{ppm})$; ${\delta \mathit{iso}}_{,\mathit{water}}=-0.011(\frac{\mathit{ppm}}{K})T+3.03(\mathrm{ppm})$. The uncertainties are the stand error of regression slope, ±0.04 ppm/K and ±0.1 × 10⁻² ppm/K for TmDTOP and water respectively. The error bars in both x and y dimensions for each data point are on the magnitude of 1 × 10⁻³. The ratio of slope and FWHM for TmDOTP and water are 0.7 ± 0.3 and 0.09 ± 0.01 respectively. The expansion of water chemical shift vs. calibrated temperature is shown in supplementary information (Fig. S1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Sample temperature calculated from the chemical shift of the H₆ proton of TmDOTP as a function of spinning frequency. T₀ is the temperature at zero spinning asymptote. TmDOTP buffer (red open circle) and KcsA proteoliposome samples (blue open square) were characterized to demonstrate that the MAS induced heating is similar for aqueous samples. Data for the TmDOTP buffer were fit to a second order polynomial function (red dash line): $T=67\frac{\mathit{mK}}{{\mathit{Hz}}^2}{\nu }_r^2-148\frac{\mathit{mK}}{\mathit{Hz}}{\nu }_r+65\mathit{mKT}$. Data for the 1.3 mm probe is shown in the supplementary information (Fig. S2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

KcsA proteoliposome spectra with and without 25 mM TmDOTP. (A) Overlay of 2D ¹³C-¹³C correlation spectra of KcsA (blue) and KcsA with 25 mM TmDOTP (red) both in DOPE/DOPG (3:1) liposomes at pH7.5. Spectral regions containing KcsA selectivity filter marker peaks are highlighted and shown in (B) V76 Cβ-Cγ (C) T74 Cα-Cγ and T75 Cα-Cγ (D) T74 Cβ-Cα and T75 Cβ-Cα. The data suggest no significant changes in protein structure and conformation state, and no changes in spectral quality with the addition of 25 mM TmDOTP. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

(A) The temperature increase indicated by the TmDOTP chemical shift is linearly dependent on the RF pulse length (field strength = 91 kHz, duty cycle = 2.8%). Data were fit using a linear least square analysis (ΔT = 0.208τ₁−0.105). (B) The dependence on CW power amplitude is shown (τ₁ = 30 ms and duty cycle = 2.8%). Data were fit using linear least square function (ΔT = 0.037P + 0.032), P represents RF power. (C) The dependence on duty cycle percentage is shown (field strength = 91 kHz and τ₁ = 30 ms, τ₂ = 5 ms); in these experiments the duty cycle is varied by varying the recycle delay from 0.5 s to 15 s. Data were fit using linear least square analysis (ΔT = 2.860D + 0.375). D represents the duty cycle percentage. All data were collected on a KcsA proteoliposome sample with 20 mM TmDOTP and a 3.2 mm E-free probe on 900 MHz. The spinning frequency was 5 kHz with sample temperature of 276.5 ± 0.1 K (VT gas temperature was 275 K).

Fig. 7.

(A) The H₆ NMR spectra of the proteoliposome sample with 20 mM TmDOTP at neutral pH during different RF frequencies. The VT gas temperature was 275 K. The chemical shift of H₆ is reported relative to that with the decoupling pulse off. Conveniently, TmDOTP has a slope of 1.06 ± 0.04 ppm/K. (B) The sample temperature changes reported by the peak 1 and peak 2 from TmDOTP proton chemical shifts are plotted as a function of the RF power. Data were collected on the 3.2 mm E-free probe at 900 MHz.