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

Zhang, Dongyu; Itin, Boris; McDermott, Ann E.

doi:10.1016/j.jmr.2019.106574

Cited by 12 publications

(12 citation statements)

References 31 publications

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“…This approach is consistent with the experimental data presented in this study and is justified by fast rotation of the amino group‐ about the C(2)–N(2) bond in our experimental conditions. It is worth noting that considerable heating may occur in samples spinning at ν R >60 kHz due to friction . Therefore our data is also consistent with Adrian et al ., who reported a coalescence temperature of ca.…”

Section: Resultssupporting

confidence: 92%

Self‐Assembly of DNA and RNA Building Blocks Explored by Nitrogen‐14 NMR Crystallography: Structure and Dynamics

2020

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The isotopic enrichment of nucleic acids with nitrogen-15 is often carried out by solid-phase synthesis of oligonucleotides using phosphoramidite precursors that are synthetically demanding and expensive. These synthetic challenges, combined with the overlap of chemical shifts, explain the lag of nitrogen-15 NMR studies of nucleic acids behind those of proteins. For the structural characterization of DNA and RNA-related systems, new NMR methods that exploit the naturally occurring 99.9 % abundant nitrogen-14 isotope are therefore highly desirable. In this study, we have investigated nitrogen-14 spectra of selfassembled quartets based on the nucleobase guanine in the solid state by means of magic-angle spinning NMR spectro-scopy. The network of dipolar proton-nitrogen couplings between neighboring stacked purine units is probed by 2D spectra based on 1 H! 14 N! 1 H double cross-polarization. Interplane dipolar contacts are identified between the stacked G quartets. The assignment is supported by density functional theory (DFT) calculations of the anisotropic chemical shifts and quadrupolar parameters. The experimental spectra are fully consistent with internuclear distances obtained in silico. Averaging of chemical shifts due to internal motions can be interpreted by semiempirical calculations. This method can easily be extended to synthetic G quartets based on nucleobase or nucleoside analogs and potentially to oligonucleotides.[a] Dr.

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Section: Resultssupporting

confidence: 92%

Self‐Assembly of DNA and RNA Building Blocks Explored by Nitrogen‐14 NMR Crystallography: Structure and Dynamics

2020

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“…Experiments at 900 MHz were collected with a spinning frequency, ω r /2 π , of 16 666 ± 10 Hz and a set point temperature of 267 ± 1 K. The sample temperature is estimated to be 290 ± 5 K due to sample heating from magic angle spinning (MAS) and RF pulses. 33 Standard π /2 pulse lengths of 2.55, 4, and 5.1 µ s were used for the 1 H, 13 C, and 15 N channels, respectively, corresponding to ω 1 /2 π = 98 kHz ( 1 H), 62.5 kHz ( 13 C), and 49 kHz ( 15 N). Two-dimensional (2D) 13 C– 13 C, 2D 13 C– 15 N (NcaCX, NcoCX), and three-dimensional (3D) 13 C– 13 C– 15 N (NCOCX, NCACX) correlation experiments were used to determine the chemical shift assignments.…”

Section: Methodsmentioning

confidence: 99%

“…Experiments at 750 MHz were collected with spinning frequencies of 16 000 ± 10 or 33 333 ± 10 Hz and a set point temperature of 230 ( ω r /2 π = 33 333 Hz) or 240 ( ω r /2 π = 16 000 Hz) ± 1 K. The sample temperature is estimated to be 290 ± 5 K due to sample heating from MAS and RF pulses. 33 Standard π /2 pulse lengths of 2.55, 3.3, and 4.3 µ s were used for the 1 H, 13 C, and 15 N channels, respectively, corresponding to ω 1 /2 π = 98 kHz ( 1 H), 76 kHz ( 13 C), and 58 kHz ( 15 N). The same 3D experiments for chemical shift assignments were performed at 750 MHz.…”

Section: Methodsmentioning

confidence: 99%

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel

Pérez-Conesa

Keeler

Zhang

et al. 2021

The Journal of Chemical Physics

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As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

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“…Experiments at 750 MHz were collected with spinning frequencies of 16000 ± 10 or 33333 ± 10Hz and a set point temperature of 230 (ω r /2π = 33333Hz) or 240 (ω r /2π = 16000Hz) ± 1 K. The sample temperature is estimated to be 290 ± 5 K due to sample heating from MAS and RF pulses. 33 Standard π/2 pulse lengths of 2.55 μs, 3.3 μs, and 4.3 μs were used for the 1 H, 13 C, and 15 N channels, respectively, corresponding to ω 1 /2π = 98 kHz ( 1 H), 76kHz( 13 C), and 58 kHz ( 15 N). The same 3D experiments for chemical shift assignments were performed at 750 MHz.…”

Section: Methodsmentioning

confidence: 99%

“…Experiments at 900 MHz were collected with a spinning frequency, ω r /2π, of 16666 ± 10 Hz and a set point temperature of 267 ± 1 K. The sample temperature is estimated to be 290 ± 5 K due to sample heating from MAS and RF pulses. 27 Standard π/2 pulse lengths of 2.55 µs, 4 µs, and 5.1 µs were used for the 1 H, 13 C, and 15 N channels, respectively, corresponding to ω 1 /2π = 98 kHz (1H), 62.5 kHz ( 13 C), and 49 kHz( 15 N). Two-dimensional (2D) 13 C- 13 C, 2D 13 C-15 N (NcaCX, NcoCX), and three-dimensional (3D) 13 C-13 C-15 N (NCOCX, NCACX) correlation experiments were used to determine the chemical shift assignments.…”

Section: B Nmr Spectroscopymentioning

confidence: 99%

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel

Pérez-Conesa

Keeler

Zhang

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

As the first potassium channel with a X-ray structure determined, and given its homol- ogy to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related in particular to the allosteric coupling between its gates remain open. The many currently available X-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, com- plexation with antibodies, and substitution of detergents for lipids, examining the channel under more native conditions is desirable. Solid-state NMR (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the acti- vated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible X-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs. fully open ensembles from MD simulations. The simulated NMR chemical shifts for those spe- cific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discrimi- nate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

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

Cited by 12 publications

References 31 publications

Self‐Assembly of DNA and RNA Building Blocks Explored by Nitrogen‐14 NMR Crystallography: Structure and Dynamics

Self‐Assembly of DNA and RNA Building Blocks Explored by Nitrogen‐14 NMR Crystallography: Structure and Dynamics

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel

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