Category Archives: Low Temperature

Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids

Potapov, A., W.M. Yau, and R. Tycko, Dynamic nuclear polarization-enhanced (13)C NMR spectroscopy of static biological solids. J Magn Reson, 2013. 231(0): p. 5-14.

http://www.ncbi.nlm.nih.gov/pubmed/23562665

We explore the possibility of using dynamic nuclear polarization (DNP) to enhance signals in structural studies of biological solids by solid state NMR without sample spinning. Specifically, we use 2D (13)C-(13)C exchange spectroscopy to probe the peptide backbone torsion angles (varphi, psi) in a series of selectively (13)C-labeled 40-residue beta-amyloid (Abeta1-40) samples, in both fibrillar and non-fibrillar states. Experiments are carried out at 9.39T and 8K, using a static double-resonance NMR probe and low-power microwave irradiation at 264GHz. In frozen solutions of Abeta1-40 fibrils doped with DOTOPA-TEMPO, we observe DNP signal enhancement factors of 16-21. We show that the orientation- and frequency-dependent spin polarization exchange between sequential backbone carbonyl (13)C labels can be simulated accurately using a simple expression for the exchange rate, after experimentally determined homogeneous (13)C lineshapes are incorporated in the simulations. The experimental 2D (13)C-(13)C exchange spectra place constraints on the varphi and psi angles between the two carbonyl labels. Although the data are not sufficient to determine varphi and psi uniquely, the data do provide non-trivial constraints that could be included in structure calculations. With DNP at low temperatures, 2D (13)C-(13)C exchange spectra can be obtained from a 3.5mg sample of Abeta1-40 fibrils in 4h or less, despite the broad (13)C chemical shift anisotropy line shapes that are observed in static samples.

Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids

Potapov, A., W.M. Yau, and R. Tycko, Dynamic nuclear polarization-enhanced (13)C NMR spectroscopy of static biological solids. J Magn Reson, 2013. 231(0): p. 5-14.

http://www.ncbi.nlm.nih.gov/pubmed/23562665

We explore the possibility of using dynamic nuclear polarization (DNP) to enhance signals in structural studies of biological solids by solid state NMR without sample spinning. Specifically, we use 2D (13)C-(13)C exchange spectroscopy to probe the peptide backbone torsion angles (varphi, psi) in a series of selectively (13)C-labeled 40-residue beta-amyloid (Abeta1-40) samples, in both fibrillar and non-fibrillar states. Experiments are carried out at 9.39T and 8K, using a static double-resonance NMR probe and low-power microwave irradiation at 264GHz. In frozen solutions of Abeta1-40 fibrils doped with DOTOPA-TEMPO, we observe DNP signal enhancement factors of 16-21. We show that the orientation- and frequency-dependent spin polarization exchange between sequential backbone carbonyl (13)C labels can be simulated accurately using a simple expression for the exchange rate, after experimentally determined homogeneous (13)C lineshapes are incorporated in the simulations. The experimental 2D (13)C-(13)C exchange spectra place constraints on the varphi and psi angles between the two carbonyl labels. Although the data are not sufficient to determine varphi and psi uniquely, the data do provide non-trivial constraints that could be included in structure calculations. With DNP at low temperatures, 2D (13)C-(13)C exchange spectra can be obtained from a 3.5mg sample of Abeta1-40 fibrils in 4h or less, despite the broad (13)C chemical shift anisotropy line shapes that are observed in static samples.

Achievement of high nuclear spin polarization using lanthanides as low-temperature NMR relaxation agents

Peat, D.T., et al., Achievement of high nuclear spin polarization using lanthanides as low-temperature NMR relaxation agents. Phys. Chem. Chem. Phys., 2013. 15(20): p. 7586-7591.

http://www.ncbi.nlm.nih.gov/pubmed/23588269

Many approaches are now available for achieving high levels of nuclear spin polarization. One of these methods is based on the notion that as the temperature is reduced, the equilibrium nuclear polarization will increase, according to the Boltzmann distribution. The main problem with this approach is the length of time it may take to approach thermal equilibrium at low temperatures, since nuclear relaxation times (characterized by the spin-lattice relaxation time T1) can become very long. Here, we show, by means of relaxation time measurements of frozen solutions, that selected lanthanide ions, in the form of their chelates with DTPA, can act as effective relaxation agents at low temperatures. Differential effects are seen with the different lanthanides that were tested, holmium and dysprosium showing highest relaxivity, while gadolinium is ineffective at temperatures of 20 K and below. These observations are consistent with the known electron-spin relaxation time characteristics of these lanthanides. The maximum relaxivity occurs at around 10 K for Ho-DTPA and 20 K for Dy-DTPA. Moreover, these two agents show only modest relaxivity at room temperature, and can thus be regarded as relaxation switches. We conclude that these agents can speed up solid state NMR experiments by reducing the T1 values of the relevant nuclei, and hence increasing the rate at which data can be acquired. They could also be of value in the context of a simple low-cost method of achieving several-hundred-fold improvements in polarization for experiments in which samples are pre-polarized at low temperatures, then rewarmed and dissolved immediately prior to analysis.

Achievement of high nuclear spin polarization using lanthanides as low-temperature NMR relaxation agents

Peat, D.T., et al., Achievement of high nuclear spin polarization using lanthanides as low-temperature NMR relaxation agents. Phys. Chem. Chem. Phys., 2013. 15(20): p. 7586-7591.

http://www.ncbi.nlm.nih.gov/pubmed/23588269

Many approaches are now available for achieving high levels of nuclear spin polarization. One of these methods is based on the notion that as the temperature is reduced, the equilibrium nuclear polarization will increase, according to the Boltzmann distribution. The main problem with this approach is the length of time it may take to approach thermal equilibrium at low temperatures, since nuclear relaxation times (characterized by the spin-lattice relaxation time T1) can become very long. Here, we show, by means of relaxation time measurements of frozen solutions, that selected lanthanide ions, in the form of their chelates with DTPA, can act as effective relaxation agents at low temperatures. Differential effects are seen with the different lanthanides that were tested, holmium and dysprosium showing highest relaxivity, while gadolinium is ineffective at temperatures of 20 K and below. These observations are consistent with the known electron-spin relaxation time characteristics of these lanthanides. The maximum relaxivity occurs at around 10 K for Ho-DTPA and 20 K for Dy-DTPA. Moreover, these two agents show only modest relaxivity at room temperature, and can thus be regarded as relaxation switches. We conclude that these agents can speed up solid state NMR experiments by reducing the T1 values of the relevant nuclei, and hence increasing the rate at which data can be acquired. They could also be of value in the context of a simple low-cost method of achieving several-hundred-fold improvements in polarization for experiments in which samples are pre-polarized at low temperatures, then rewarmed and dissolved immediately prior to analysis.

Efficient, balanced, transmission line RF circuits by back propagation of common impedance nodes

Markhasin, E., et al., Efficient, balanced, transmission line RF circuits by back propagation of common impedance nodes. J. Magn. Reson., 2013. 231(0): p. 32-38.

http://dx.doi.org/10.1016/j.jmr.2013.02.017

We present a new, efficient strategy for designing fully balanced transmission line RF circuits for solid state NMR probes based on back propagation of common impedance nodes (BPCIN). In this approach, the impedance node phenomenon is the sole means of achieving mutual RF isolation and balance in all RF channels. BPCIN is illustrated using a custom double resonance 3.2 mm MAS probe operating at 500 MHz (1H) and 125 MHz (13C). When fully optimized, the probe is capable of producing high homogeneity (810°/90° ratios of 86% and 89% for 1H and 13C, respectively) and high efficiency (γB1 = 100 kHz for 1H and 13C at 70 W and 180 W of RF input, respectively; up to 360 kHz for 1H). The probe’s performance is illustrated by 2D MAS correlation spectra of microcrystals of the tripeptide N-f-MLF-OH and hydrated amyloid fibrils of the protein PI3-SH3.

Efficient, balanced, transmission line RF circuits by back propagation of common impedance nodes

Markhasin, E., et al., Efficient, balanced, transmission line RF circuits by back propagation of common impedance nodes. J. Magn. Reson., 2013. 231(0): p. 32-38.

http://dx.doi.org/10.1016/j.jmr.2013.02.017

We present a new, efficient strategy for designing fully balanced transmission line RF circuits for solid state NMR probes based on back propagation of common impedance nodes (BPCIN). In this approach, the impedance node phenomenon is the sole means of achieving mutual RF isolation and balance in all RF channels. BPCIN is illustrated using a custom double resonance 3.2 mm MAS probe operating at 500 MHz (1H) and 125 MHz (13C). When fully optimized, the probe is capable of producing high homogeneity (810°/90° ratios of 86% and 89% for 1H and 13C, respectively) and high efficiency (γB1 = 100 kHz for 1H and 13C at 70 W and 180 W of RF input, respectively; up to 360 kHz for 1H). The probe’s performance is illustrated by 2D MAS correlation spectra of microcrystals of the tripeptide N-f-MLF-OH and hydrated amyloid fibrils of the protein PI3-SH3.

NMR at Low and Ultralow Temperatures

Tycko, R., NMR at Low and Ultralow Temperatures. Acc. Chem. Res., 2013.

http://dx.doi.org/10.1021/ar300358z

Solid state nuclear magnetic resonance (NMR) measurements at low temperatures have been common in physical sciences for many years and are becoming increasingly important in studies of biomolecular systems. This Account reviews a diverse set of projects from my laboratory, dating back to the early 1990s, that illustrate the motivations for low-temperature solid state NMR, the types of information that are available from the measurements, and likely directions for future research. These projects include NMR studies of both physical and biological systems, performed at low (cooled with nitrogen, down to 77 K) and ultralow (cooled with helium, below 77 K) temperatures, and performed with and without magic-angle spinning (MAS). NMR studies of physical systems often focus on phenomena that occur only at low temperatures. Two examples from my laboratory are studies of molecular rotation and orientational ordering in solid C60 at low temperatures and studies of unusual electronic states, called skyrmions, in two-dimensionally confined electron systems within semiconductor quantum wells. To study quantum wells, we used optical pumping of nuclear spin polarizations to enhance their NMR signals. The optical pumping phenomenon exists only at ultralow temperatures. In studies of biomolecular systems, low-temperature NMR has several motivations. In some cases, low temperatures suppress molecular tumbling, thereby permitting solid state NMR measurements on soluble proteins. Studies of AIDS-related peptide/antibody complexes illustrate this effect. In other cases, low temperatures suppress conformational exchange, thereby permitting quantitation of conformational distributions. Studies of chemically denatured states of the model protein HP35 illustrate this effect. Low temperatures and rapid freeze-quenching can also be used to trap transient intermediate states in a non-equilibrium kinetic process, as shown in studies of a transient intermediate in the rapid folding pathway of HP35. NMR sensitivity generally increases with decreasing sample temperature. Therefore, it can be useful to carry out experiments at the lowest possible temperatures, particularly in studies of biomolecular systems in frozen solutions. However, solid state NMR studies of biomolecular systems generally require rapid MAS. A novel MAS NMR probe design that uses nitrogen gas for sample spinning and cold helium only for sample cooling allows a wide variety of solid state NMR measurements to be performed on biomolecular systems at 20?25 K, where signals are enhanced by factors of 12?15 relative to measurements at room temperature. MAS NMR at ultralow temperatures also facilitates dynamic nuclear polarization (DNP), allowing sizeable additional signal enhancements and large absolute NMR signal amplitudes with relatively low microwave powers. Current research in my laboratory seeks to develop and exploit DNP-enhanced MAS NMR at ultralow temperatures, for example, in studies of transient intermediates in protein folding and aggregation processes and studies of peptide/protein complexes that can be prepared only at low concentrations.

NMR at Low and Ultralow Temperatures

Tycko, R., NMR at Low and Ultralow Temperatures. Acc. Chem. Res., 2013.

http://dx.doi.org/10.1021/ar300358z

Solid state nuclear magnetic resonance (NMR) measurements at low temperatures have been common in physical sciences for many years and are becoming increasingly important in studies of biomolecular systems. This Account reviews a diverse set of projects from my laboratory, dating back to the early 1990s, that illustrate the motivations for low-temperature solid state NMR, the types of information that are available from the measurements, and likely directions for future research. These projects include NMR studies of both physical and biological systems, performed at low (cooled with nitrogen, down to 77 K) and ultralow (cooled with helium, below 77 K) temperatures, and performed with and without magic-angle spinning (MAS). NMR studies of physical systems often focus on phenomena that occur only at low temperatures. Two examples from my laboratory are studies of molecular rotation and orientational ordering in solid C60 at low temperatures and studies of unusual electronic states, called skyrmions, in two-dimensionally confined electron systems within semiconductor quantum wells. To study quantum wells, we used optical pumping of nuclear spin polarizations to enhance their NMR signals. The optical pumping phenomenon exists only at ultralow temperatures. In studies of biomolecular systems, low-temperature NMR has several motivations. In some cases, low temperatures suppress molecular tumbling, thereby permitting solid state NMR measurements on soluble proteins. Studies of AIDS-related peptide/antibody complexes illustrate this effect. In other cases, low temperatures suppress conformational exchange, thereby permitting quantitation of conformational distributions. Studies of chemically denatured states of the model protein HP35 illustrate this effect. Low temperatures and rapid freeze-quenching can also be used to trap transient intermediate states in a non-equilibrium kinetic process, as shown in studies of a transient intermediate in the rapid folding pathway of HP35. NMR sensitivity generally increases with decreasing sample temperature. Therefore, it can be useful to carry out experiments at the lowest possible temperatures, particularly in studies of biomolecular systems in frozen solutions. However, solid state NMR studies of biomolecular systems generally require rapid MAS. A novel MAS NMR probe design that uses nitrogen gas for sample spinning and cold helium only for sample cooling allows a wide variety of solid state NMR measurements to be performed on biomolecular systems at 20?25 K, where signals are enhanced by factors of 12?15 relative to measurements at room temperature. MAS NMR at ultralow temperatures also facilitates dynamic nuclear polarization (DNP), allowing sizeable additional signal enhancements and large absolute NMR signal amplitudes with relatively low microwave powers. Current research in my laboratory seeks to develop and exploit DNP-enhanced MAS NMR at ultralow temperatures, for example, in studies of transient intermediates in protein folding and aggregation processes and studies of peptide/protein complexes that can be prepared only at low concentrations.

Development of DNP-Enhanced High-Resolution Solid-State NMR System for the Characterization of the Surface Structure of Polymer Materials

Horii, F., et al., Development of DNP-Enhanced High-Resolution Solid-State NMR System for the Characterization of the Surface Structure of Polymer Materials. J. Infrared Millim. Te., 2012. 33(7): p. 756-765.

http://dx.doi.org/10.1007/s10762-012-9874-1

A dynamic nuclear polarization (DNP)-enhanced cross-polarization/magic-angle spinning (DNP/CP/MAS) NMR system has been developed by combining a 200 MHz Chemagnetics CMX-200 spectrometer operating at 4.7 T with a high-power 131.5 GHz Gyrotron FU CW IV. The 30 W sub-THz wave generated in a long pulse TE $ _{{41}}^{{(1)}} $ mode with a frequency of 5 Hz was successfully transmitted to the modified Doty Scientific low-temperature CP/MAS probe through copper smooth-wall circular waveguides. Since serious RF noises on NMR signals by arcing in the electric circuit of the probe and undesired sample heating were induced by the continuous sub-THz wave pulse irradiation with higher powers, the on-off sub-THz wave pulse irradiation synchronized with the NMR detection was developed and the appropriate setting of the irradiation time and the cooling time corresponding to the non-irradiation time was found to be very effective for the suppression of the arcing and the sample heating. The attainable maximum DNP enhancement was more than 30 folds for C1 13 C-enriched D -glucose dissolved in the frozen medium containing mono-radical 4-amino-TEMPO. The first DNP/CP/MAS 13 C NMR spectra of poly(methyl methacrylate) (PMMA) sub-micron particles were obtained at the dispersed state in the same frozen medium, indicating that DNP-enhanced 1 H spins effectively diffuse from the medium to the PMMA particles through their surface and are detected as high-resolution 13 C spectra in the surficial region to which the 1 H spins reach. On the basis of these results, the possibility of the DNP/CP/MAS NMR characterization of the surface structure of nanomaterials including polymer materials was discussed.

Development of DNP-Enhanced High-Resolution Solid-State NMR System for the Characterization of the Surface Structure of Polymer Materials

Horii, F., et al., Development of DNP-Enhanced High-Resolution Solid-State NMR System for the Characterization of the Surface Structure of Polymer Materials. J. Infrared Millim. Te., 2012. 33(7): p. 756-765.

http://dx.doi.org/10.1007/s10762-012-9874-1

A dynamic nuclear polarization (DNP)-enhanced cross-polarization/magic-angle spinning (DNP/CP/MAS) NMR system has been developed by combining a 200 MHz Chemagnetics CMX-200 spectrometer operating at 4.7 T with a high-power 131.5 GHz Gyrotron FU CW IV. The 30 W sub-THz wave generated in a long pulse TE $ _{{41}}^{{(1)}} $ mode with a frequency of 5 Hz was successfully transmitted to the modified Doty Scientific low-temperature CP/MAS probe through copper smooth-wall circular waveguides. Since serious RF noises on NMR signals by arcing in the electric circuit of the probe and undesired sample heating were induced by the continuous sub-THz wave pulse irradiation with higher powers, the on-off sub-THz wave pulse irradiation synchronized with the NMR detection was developed and the appropriate setting of the irradiation time and the cooling time corresponding to the non-irradiation time was found to be very effective for the suppression of the arcing and the sample heating. The attainable maximum DNP enhancement was more than 30 folds for C1 13 C-enriched D -glucose dissolved in the frozen medium containing mono-radical 4-amino-TEMPO. The first DNP/CP/MAS 13 C NMR spectra of poly(methyl methacrylate) (PMMA) sub-micron particles were obtained at the dispersed state in the same frozen medium, indicating that DNP-enhanced 1 H spins effectively diffuse from the medium to the PMMA particles through their surface and are detected as high-resolution 13 C spectra in the surficial region to which the 1 H spins reach. On the basis of these results, the possibility of the DNP/CP/MAS NMR characterization of the surface structure of nanomaterials including polymer materials was discussed.

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