New 1.2 GHz NMR Spectrometers - New Horizons?

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  • Published: Oct 3, 2017
  • Author: Prof. Dr. Harald Schwalbe
  • Source: Angewandte Chemie
  • Channels: NMR Knowledge Base / MRI Spectroscopy
thumbnail image: New 1.2 GHz NMR Spectrometers - New Horizons?

Article reproduced from: Angewandte Chemie, 2017, 56, 10252-10253

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Building ultrahigh-field NMR spectrometers with magnetic strengths of 1.2 GHz (28.2 T) that cost more than 12.5 million Euros poses two challenges. At 5x105 times the earth's magnetic field, the strength of the target magnetic field is enormous. Furthermore, the field in the magnet's active volume, a zone of only around 1 cm3, must be extremely homogenous, that is 99.99999999 % of the active volume must have exactly the same magnetic field strength and maintain this homogeneity without being impacted by external influences.

The new magnet technology has been developed by the company Bruker. It represents both a scientific breakthrough and an act of impressive technological innovation. The entire magnet design had to be changed, replacing the currently used Nb3Sn-based low-temperature superconducting (LTS) wire technology with a hybrid design, as the critical current of Nb3Sn, the most advanced “classical” low-temperature superconductor, drops to zero in the vicinity of a magnetic field strength of 23.5 T. Rare-earth–barium–copper oxides (REBCO; with REs such as yttrium), high-temperature superconductors (HTSs), were found to be particularly well suited for that purpose. REBCO superconductors are available as stainless steel tapes coated with a layer of YBCO. The new materials pose several challenges: The tapes have a width of a few millimeters, which makes it more difficult to obtain homogeneous fields. In addition, commercially available HTS tapes are several hundred meters long, whereas building 1.2 GHz NMR magnets requires a wire longer than a kilometer.

Only two years ago, a Review published in this journal (J. H. Ardenkjaer-Larsen et al., Angew. Chem. Int. Ed. 2015, 54, 9162) came to the conclusion that there was only a very small chance that hybrid magnets combining two different wire types could be built and operated in persistent mode. The authors predicted that hybrid magnets would need constant field charging and correcting to make them sufficiently stable for cutting-edge NMR applications. Bruker now has successfully assembled a high-field LTS–HTS hybrid magnet and plans to assemble the first 1.1 GHz magnet this year and to deliver the first 1.2 GHz magnets next year. Orders have been placed for 1.2 GHz spectrometers by laboratories from (in alphabetical order) Florence, Frankfurt, Göttingen, Jülich, Lille, Munich, and Utrecht.

With NMR spectroscopy, monomers or polymers can be analyzed alike, whether chemically synthesized, biochemically produced, or isolated from natural samples, even including paleontological sources. NMR spectroscopy is a noninvasive method that does not require molecules to be in a particular state and that can be applied even to molecules in cells. Because of its enormous range of applications, NMR spectroscopy continues to play an important role in very different research areas.

In the past, every new generation of high-field NMR spectrometers provided a major impulse for NMR spectroscopy. We must keep in mind, however, that the introduction of advanced equipment requires large investments that have to be justified. National funding agencies need coordinated funding strategies to set aside the required budgets. Scientists for their part have to explain very clearly what results the new technology might yield, that could not be obtained at lower field strengths. In doing so it helps to reflect on past experience and extrapolate to the future. The sensitivity of detection goes up at least with B03/2, and the resolution in n-dimensional experiments with B0n. As we now even record 7D experiments, the increases in sensitivity and resolution that arise from higher field strengths will benefit many areas of research.

In the early 1990s, 600 MHz NMR spectrometers became available. They paved the way for studying proteins and molecular biology methods became available for enriching proteins with the rare 15N and 13C isotopes. At the same time, multidimensional NMR methods were developed that allowed full exploitation of the rich information content of all protein nuclei except for oxygen and sulfur in spectra, yielding thousands of chemical shifts as well as J couplings and NOEs between atom pairs. Based on this information, around 10 % of such macromolecular structures have been determined by NMR spectroscopy. Furthermore, NMR is the only method with which local multistate conformational equilibria and their interconversion kinetics can be characterized with atomic resolution.

Then came the 800 MHz NMR spectrometers and the possibility to detect the long predicted partial residual alignment of biomolecules, in particular of DNA and paramagnetic proteins. The degree of alignment scales with B02; therefore, higher fields were required to detect the small effects. The concept of extracting long-range orientational restraints to discover dynamics of motion had been proposed before, but 800 MHz spectrometers were needed to determine residual dipolar couplings (RDCs) as differences in multiplet splittings recorded at the highest and a lower field. This observation provided the necessary confidence to embark on this path to develop further methods to align molecules.

The introduction of 900 MHz spectrometers then facilitated the discovery of the TROSY effect. TROSY tore down the size-limitation boundaries of liquid-state NMR spectroscopy as it allows us to use NMR spectroscopy for the investigation of (symmetric) megadalton protein complexes such as the proteasome (protein degradation machine) or the molecular chaperone GroEL. Follow-up strategies were developed and other very slowly decaying coherences were detected, in particular for methyl groups. At the same time, fast spinning at the magic angle combined with high magnetic-field strength (making dipolar couplings weaker) triggered a resolution revolution in solid-state NMR spectroscopy.

I will now try to answer the question about how the increasingly large budgets needed for next-generation NMR spectrometers can be justified, and describe the potential scientific progress that 1.2 GHz spectrometers might bring, even at the risk of being proven wrong in the future by breakthroughs in totally different areas of NMR.

Both liquid-state and solid-state NMR will benefit from the new technology. In liquid-state NMR, the increasingly high magnetic fields will allow us to isolate increasing numbers of conformational states of biomolecules and to put conformational transitions between those states into slow exchange such that separate peaks can be more readily detected. Investigations of fast exchange systems between different states will profit from a high field strength since they rely on the measurement of squared chemical shift differences. Up to now, the typical approach has been to prepare a single, homogeneous conformational state of a protein or a nucleic acid, but it is increasingly recognized that the heterogeneity of states including their interconversion kinetics is at the heart of biological functions. The discovery of excited states in RNA, DNA and proteins by relaxation dispersion and CEST technologies may serve as an example. Translational and transcriptional RNA regulation elements are another important object of investigation.

The NMR study of systems involved in protein folding and misfolding profits from complementary investigation by techniques developed for liquid- or solid-state samples. NMR has unique roles in studying intrinsically disordered regions that play crucial roles in the regulation of numerous cellular processes. In the new field of biological phase separation and membraneless organelles, NMR will be the prime method to study these ensembles that have characteristics between liquids and gels.

NMR spectroscopy will continue to be the method of choice to obtain information on dynamics on such macromolecular complexes in solution that is difficult to obtain by X-ray crystallography or cryo-EM single-particle analysis. For these assemblies, NMR data will provide molecular movies of the dynamic process and thus tell us about those dynamic regulatory regions that remain invisible to other techniques.

X-ray crystallography of G-protein coupled receptors (GPCRs), the pharmacologically most important membrane proteins, has fundamentally and impressively changed our understanding of GPCR function. Liquid- and solid-state NMR, however, will become the prime tool to investigate agonistic and antagonistic modulators of signaling mediated by GPCRs and to detect functional important intermediate states of GPCRs. Structure-based drug design will greatly benefit from highest-field NMR, in particular in providing insights into allosteric effects and conformational dynamics linked to drug binding. Metabolic flux analysis, or real-time tracer-based analysis of metabolism, at (near-) physiological conditions may serve as an ultimate example for the noninvasive characterization of unique information linked to human health and disease.

The availability of 1.2 GHz NMR spectrometers will also stimulate methods research to increase the signal-to-noise ratio at ultrahigh fields, including in liquids. New probe technologies and new combinations of NMR and other biophysical methods will follow. It is somewhat ironic that the development of high-field spectrometers also expands the range of magnetic fields that are available for studying the field dependence of relaxation effects. A combination of two scenarios might also be beneficial: shuttling NMR samples between a low-field section for magnetization transfer and a high-field section of the spectrometer for detection.

NMR is not the only structural biology technique undergoing revolutionary changes. Findings triggered by the development of free-electron laser crystallography (XFEL) and by new detectors for cryo-EM single particle and tomography analyses are impressive. Germany reacted by providing funding for the European XFEL installation in Hamburg, including four 1.2 GHz NMR spectrometers and for several cryo-EM machines. These funding decisions came at the right moment. It is important to note that all of the initiatives in structural biology have a national as well as a European dimension. NMR centers in Florence, Utrecht and Frankfurt will provide access to the new 1.2 GHz spectrometers for researchers from all over the European Union. Given the current isolationist movements, it will always be important to link national and European, if not global research endeavors, for the benefit of fundamental and applied research alike.

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Prof. Dr Harald Schwalbe is Professor of Organic Chemistry at Centre for Biomolecular Magnetic Resonance at the University of Frankfurt.

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