3

High Magnetic Fields in
Chemistry, Biochemistry,
and Biology

High-field magnets that are near the cutting edge of technology play central roles in chemical, biochemical, and biological research, primarily through the techniques of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and Fourier-transform ion cyclotron resonance (FT-ICR). In medical research and clinical medicine, high-field magnets are essential components of magnetic resonance imaging (MRI) systems, which create three-dimensional (3D) images of anatomical and diagnostic importance from NMR signals. (MRI is described in a separate section below.) In all of these techniques, current magnetic field strengths are somewhat below the level that is achieved in specialized high-field facilities devoted primarily to physics and materials research. The magnets are usually produced by commercial vendors rather than by research teams. Despite the commercial context of their construction, the magnets used for very high field NMR and other spectroscopies present ambitious design challenges: The magnets have exceptionally high homogeneity (~1 ppb) over large volumes (>1 cm3 homogeneous volume), requiring highly specialized and sophisticated engineering to manage the concomitant structural stresses and stored energies. The magnets must also have exceptionally high stability for indefinite time periods (months to years), implying that they are typically constructed from persistent superconducting materials. Field strengths in NMR magnets are limited by the properties of these materials, making high-field NMR one of the important scientific drivers for the continuing development of advanced superconducting materials and magnet technology.

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3 High Magnetic Fields in Chemistry, Biochemistry, and Biology High-field magnets that are near the cutting edge of technology play central roles in chemical, biochemical, and biological research, primarily through the techniques of nuclear magnetic resonance (NMR), electron paramagnetic reso- nance (EPR), and Fourier-transform ion cyclotron resonance (FT-ICR). In medi- cal research and clinical medicine, high-field magnets are essential components of magnetic resonance imaging (MRI) systems, which create three-dimensional (3D) images of anatomical and diagnostic importance from NMR signals. (MRI is described in a separate section below.) In all of these techniques, current magnetic field strengths are somewhat below the level that is achieved in specialized high- field facilities devoted primarily to physics and materials research. The magnets are usually produced by commercial vendors rather than by research teams. Despite the commercial context of their construction, the magnets used for very high field NMR and other spectroscopies present ambitious design challenges: The mag- nets have exceptionally high homogeneity (~1 ppb) over large volumes (>1 cm3 homogeneous volume), requiring highly specialized and sophisticated engineering to manage the concomitant structural stresses and stored energies. The magnets must also have exceptionally high stability for indefinite time periods (months to years), implying that they are typically constructed from persistent superconduct- ing materials. Field strengths in NMR magnets are limited by the properties of these materials, making high-field NMR one of the important scientific drivers for the continuing development of advanced superconducting materials and magnet technology. The National High Magnetic Field Laboratory (NHMFL) includes user facilities 67

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68 High Magnetic Field Science and I t s A pp l i c a t i o n in the US for NMR spectroscopy and FT-ICR, with state-of-the-art, but not unique, equip- ment. In addition, NHMFL has unique facilities based on magnet systems designed and built in-house. These include an ultra-wide-bore superconducting 900 MHz NMR magnet, which allows MRI studies on animals that are not possible elsewhere; high-field FT-ICR systems (including a 21 T system currently under construction through a joint effort with a commercial vendor), and high-field EPR systems. The high-field hybrid magnets and resistive magnets of NHMFL have also been used for NMR and EPR studies, principally of nonbiological systems to date. It is impossible to overstate the importance of NMR as an analytical and structural tool in chemistry. When chemists synthesize new compounds with potential applications in medicine or technology, they always use NMR measure- ments to determine the chemical structure of these compounds and to optimize the synthetic approach. In the biological sciences, NMR measurements are one of the two main tools by which scientists determine full three-dimensional structures of proteins and nucleic acids, the other being X-ray crystallography. In materials science, NMR provides essential information not only about structure, but also about the electronic and magnetic properties that determine technological useful- ness. For paramagnetic systems, including enzymes and supramolecular complexes that are crucial for numerous biological processes and materials that are important in industrial catalysis and energy storage, EPR measurements provide additional chemical, structural, and mechanistic information that cannot be obtained from NMR, crystallography, or other methods. In both chemistry and biochemistry, FT-ICR permits the determination of molecular masses with the highest available precision and accuracy, especially for the complex chemical mixtures that occur in such diverse fields as petroleum research, proteomics, and metabolomics. Principles of NMR and Importance of High Fields in NMR Nuclei of certain atomic isotopes, including 1H, 13C, 15N, 17O, 29Si, and 31P, possess intrinsic angular momenta, called “nuclear spin,” and associated magnetic moments. In an external magnetic field, interaction between nuclear magnetic moments and the external field causes the nuclei to align with the field direction. NMR is a phenomenon in which the application of radio-frequency (RF) pulses induces a precession of the aligned nuclei about the external field direction, in turn resulting in the emission of RF signals at the characteristic precession frequen- cies. The precise frequencies and amplitudes of the emitted signals constitute the NMR spectrum. Although the basic phenomenon of NMR was demonstrated and explained over 60 years ago, the utility of NMR spectra in the physical, chemical, and biological sciences expanded substantially in each subsequent decade. The continuing expansion of NMR into new areas of science is a result of the fact that the details of NMR spectra are exquisitely sensitive to the chemical, structural,

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High Magnetic Fields in Chemistry, Biochemistry, and Biology 69 electronic, and motional properties of molecules and materials. A steady stream of advances in NMR methods and instrumentation, including but not limited to higher fields, has allowed NMR measurements to be performed on a progressively larger variety of systems with increasing complexity. Higher magnetic fields lead to better NMR data for two main reasons. The first is spectral resolution: The NMR frequency of the nucleus of a particular atom in a molecule or material is proportional to the strength of the external field but is also affected by the atom’s local chemical and structural environment. As the external field increases, differences between NMR frequencies of different atoms become proportionally larger and easier to measure. One of the most important advances in modern NMR methodology, beginning in the mid-1970s, is the development of “multidimensional” NMR spectroscopy, in which NMR frequencies detected in multiple time periods within a single RF pulse sequence are correlated with one another. In an N-dimensional NMR spectrum, the effect of increasing magnetic field on spectral resolution occurs in each dimension, so that the number of distinct NMR frequencies that can be measured (which determines the size and complexity of molecules and materials that can be studied by NMR) can increase as roughly the Nth power of the magnetic field strength (BN). In practice, in a 3D NMR spectrum of a biological macromolecule such as a protein in aqueous solution in a field of approximately 20 T, NMR signals from more than 10,000 1H, 13C, and 15N nuclei can be resolved from one another and measured accurately. The second main reason why higher fields lead to better NMR data is sensitiv- ity: In available magnets, NMR frequencies typically lie in the 100-1000 MHz range, corresponding to photon energies of 4 × 10−7 to 4 × 10−6 eV (5-50 mK). These low energies imply that the degree of nuclear alignment induced by the magnetic field (i.e., the fractional difference between nuclear spin momenta parallel and antiparallel to the field direction, called the nuclear spin polarization) is typically only 10−6-10−5 at ambient temperature and is proportional to the field strength. NMR signal amplitudes are proportional to the nuclear spin polarization. Because NMR signals are detected inductively, the signal amplitudes are also proportional to NMR frequencies themselves. Thus, signal-to-noise ratios in NMR spectra can be proportional to B2. Additional factors affecting sensitivity include the tempera- tures of samples and electronics, the NMR linewidths, and the repetition rates of measurements (which are limited by spin-lattice relaxation rates, the rates at which nuclei align with the magnetic field before each measurement). In practice, an approximately linear dependence of NMR sensitivity on magnetic field strength is often observed. This produces an approximately linear decrease in sample quanti- ties required for NMR measurements, an important consideration especially for biological samples that are difficult to obtain in large quantities. Two distinct classes of NMR techniques are important in studies of chemical, biochemical, and biological systems. In each class, higher fields produce additional

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70 High Magnetic Field Science and I t s A pp l i c a t i o n in the US advantages for distinct reasons. The most common techniques, called “solution NMR,” apply to molecules that are dissolved in an isotropic liquid (e.g., aqueous buffers or organic solvents). Rapid translational and rotational diffusion in an isotropic liquid make all molecules in the sample structurally equivalent on the nanosecond-to-microsecond timescale. Rapid rotational diffusion also averages out anisotropic nuclear spin interactions, resulting in exceptionally narrow NMR lines and high spectral resolution. However, when molecules become very large, as in the case of high-molecular-weight proteins and nucleic acids, rotational diffusion becomes too slow, resulting in greater linewidths that impair both resolution and sensitivity (because the NMR linewidths limit the efficiency of nuclear spin polar- ization transfers that are essential for multidimensional spectroscopy). However, in certain cases, higher fields reduce the NMR linewidths of high-molecular-weight proteins and nucleic acids through a partial cancellation between linewidth con- tributions from anisotropic magnetic dipole-dipole interactions, which are inde- pendent of field, and anisotropic chemical shielding interactions, which increase linearly with field. Thus, in the case of biologically important macromolecules in solution, higher fields enable multidimensional NMR measurements on high- molecular-weight systems that would otherwise be impossible. Very high fields can also produce a weak magnetic alignment of dissolved molecules, due to anisotropy in their magnetic susceptibility, which leads to incomplete averaging of dipole- dipole interactions among nuclei. Solution NMR measurements of these residual dipole-dipole interactions provide useful constraints on molecular structures, as has been demonstrated for proteins. The second class of NMR techniques, called “solid state NMR,” applies to bona fide solids, either crystalline or noncrystalline, that are of interest in materials sci- ence and organic and inorganic chemistry, as well as to solidlike biochemical and biological systems, including protein filaments and membrane-associated systems. The absence of isotropic translational and rotational diffusion (and, particularly at low temperatures, the absence of internal molecular motions) in a solid typi- cally results in significantly greater NMR linewidths and poor spectral resolution. However, the technique of magic-angle spinning (MAS), first demonstrated in the late 1950s and improved dramatically in recent years, in which solid samples are rotated very rapidly about an axis at the “magic angle” qM = cos−1(1/√3) to the magnetic field direction using a pneumatic turbine system, approximates the effects of rotational diffusion, producing solid state NMR linewidths that can approach the linewidths in solution NMR spectra. Some of the most exciting applications of solid-state NMR are possible only at very high magnetic fields. In solid-state NMR of organic and biological systems, strong dipole-dipole interactions among 1H nuclei limit the achievable 1H NMR linewidths, even under rapid MAS. Therefore, it is only at the highest available fields that 1H NMR spectra of complex organic and biological systems become useful. Inorganic systems of practical and chemical

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High Magnetic Fields in Chemistry, Biochemistry, and Biology 71 interest (e.g., catalysts, glasses, battery materials) prominently contain elements whose NMR spectra are difficult or impossible to measure at low fields, because the nuclei have spin quantum numbers greater than ½ (e.g., 7Li, 17O, 27Al). These nuclei possess electric quadrupole interactions, which are averaged out to lowest order by MAS but make a second-order contribution to the NMR linewidths that is inversely proportional to the magnetic field strength. For these reasons, NMR spectra of many technologically important materials are useful only if very high field equipment is used, and are increasingly informative as the field increases. Recent Trends and Achievements in NMR In studies of biological systems, NMR is one of the two major types of mea- surements that can be used to reveal the full 3D molecular structures of macro- molecules, especially proteins and nucleic acids, the other being X-ray diffraction measurements on single crystals. In addition to purely structural information, NMR measurements have the unique capability of providing detailed, site-specific information about molecular motions in macromolecules, including motions that are essential for biological function. While X-ray diffraction measurements are largely restricted to highly structurally ordered molecules in crystalline environ- ments, NMR methods are applicable to proteins and nucleic acids in fluid environ- ments that more closely resemble the cytoplasmic and membrane environments of cells. Perturbations of NMR signals due to intermolecular interactions are used in the screening of molecular libraries for binding to pharmaceutically important macromolecular targets, providing an efficient approach to the identification of new lead compounds in drug development. NMR methods are also applicable to molecules that are intrinsically disordered, resistant to crystallization, and (in the case of solid-state NMR) inherently noncrystalline and insoluble. Since the first demonstrations of protein structure determination by NMR in the early 1980s, methodological and technological advances have contributed to a steady increase in the size and diversity of systems that can be characterized by NMR. Among these are the development of multidimensional NMR techniques that allow NMR frequencies of essentially all 1H, 15N, and 13C nuclei within a pro- tein or nucleic acid to be measured and assigned to specific atoms; the identification and characterization of a variety of nuclear spin interactions that can be measured through NMR signals and interpreted as experimental constraints on molecular structure; and the development of highly stable and homogeneous superconduct- ing magnets with fields up to 23.5 T. Some of the most significant new trends in biomolecular NMR that have appeared since the NRC report Opportunities in High Magnetic Field Science (NRC, 2005) include these:

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72 High Magnetic Field Science and I t s A pp l i c a t i o n in the US • Continued advances in the solution NMR methods for determining struc- ture and dynamics, and integration of solution NMR measurements with measurements that provide complementary structural information, especially small-angle X-ray and neutron-scattering measurements. Multidimensional solution NMR measurements are particularly powerful for obtaining short-range structural constraints that define the molecular structures of individual protein domains and specific interfaces between subunits within a supramolecular complex, while small-angle scattering data pro- vide information about the overall configuration of a multidomain protein or multisubunit complex. Long-range structural constraints can also be obtained from EPR measurements, as described below, and from electron microscopy. As an example, by combining extensive NMR data sets with small-angle X-ray scattering data, NMR spectroscopists have recently suc- ceeded in determining the complete 3D structure of an essential bacterial enzyme that exists as a homodimer, comprised of 1,148 amino acids, or nearly 18,000 atoms (Takayama et al., 2011). From a combination of NMR and cryo-electron microscopy measurements, NMR spectroscopists have determined the complete 3D structure of a large RNA structural motif, comprised of 131 nucleotide units or nearly 4,250 atoms, which is critical for packaging within retroviruses, of which HIV-1 is an example (Miyazaki et al., 2010). • Accelerated growth of biomolecular solid-state NMR. Since solid-state NMR measurements are not limited by molecular rotational diffusion rates, solu- bility, or crystallinity, these measurements have the potential to address structural and dynamical problems in important classes of systems that are not amenable to any other techniques. The full potential of biomolecu- lar solid-state NMR has begun to be realized only recently, in large part due to technological advances. Improvements in MAS technology allow sample rotation frequencies above 50 kHz to be achieved routinely; solid state NMR probes (the devices that contain the circuitry for application of RF pulses and detection of NMR signals) that work efficiently at the high NMR frequencies of high-field magnets have been developed; new isotopic labeling approaches have been introduced that lead to tractable solid-state NMR spectra for large proteins; new techniques for assigning solid-state NMR signals to specific atomic sites and for obtaining molecular structural constraints have been developed. In addition, a rapidly growing community of solid-state NMR spectroscopists has explored an increasing variety of biologically important systems. Significant achievements include the determination of complete molecular structures of filamentous pro- tein assemblies, called amyloid fibrils, that are associated with Alzheimer’s

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High Magnetic Fields in Chemistry, Biochemistry, and Biology 73 disease and related diseases and with the transmissible protein-encoded biological states known as “prions” (Paravastu et al., 2008; Wasmer et al., 2008). Solid-state NMR measurements have also provided important new structural and mechanistic information about proteins that form ion chan- nels in cell membranes (Bhate et al., 2010), and proteins that are involved in influenza infectivity and transmission (Cady et al., 2010). • Extension of NMR measurements to intact cells and subcellular structures. Proteins and other macromolecules account for roughly 20 percent of the volume within cells, creating a highly congested, heterogeneous environ- ment in which molecular structures and intermolecular interactions can in principle be significantly perturbed from their states in the simplified conditions of traditional in vitro studies. Improvements in the sensitivity and resolution of NMR are facilitating attempts to quantify the influence of the biological environment, through direct NMR measurements on proteins within bacteria or bacterial membranes (Renault et al., 2012; Fu et al., 2011). A major new trend in both solution NMR and solid state NMR is the exploita- tion of dynamic nuclear polarization (DNP) for sensitivity enhancements. DNP is a process in which the large polarizations of electron spins in a strong magnetic field are partially transferred to nuclear spins by irradiation of EPR transitions, result- ing in large enhancements of nuclear spin polarizations and hence NMR signals. Recent work has shown that NMR signal enhancements by a factor of more than 100 can be achieved through DNP in a variety of chemical and biochemical samples that are paramagnetically doped with stable free radical compounds. Among other applications, these signal enhancements have enabled new studies of metabolic and enzymatic pathways in cell cultures and whole organisms (Meier et al., 2012; Menichetti et al., 2012), have allowed solid state NMR studies of the structure and chemistry of catalyst surfaces (Lelli et al., 2011), and promise to enable structural studies of membrane-bound peptide/protein complexes that are available only in nanomole quantities (Reggie et al., 2011), including hormone/receptor complexes that are important pharmaceutical targets. In recent years, NMR has developed a new role as one of the primary experi- mental tools in the burgeoning field of metabolomics, a term that encompasses efforts to identify and quantify all small-molecule metabolites within cells, tissue, and biological fluids and to correlate variations in metabolite profiles with gene expression, disease state, and environmental factors. The high resolution and high sensitivity of NMR at high fields allows ~100 compounds with ~10 mM concen- trations to be quantified simultaneously within a single specimen (Psychogios et al., 2011).

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74 High Magnetic Field Science and I t s A pp l i c a t i o n in the US In solid state chemistry and materials chemistry, NMR investigations of materi- als designed for energy storage applications have been an active area of research, including materials for fuel cells (Buannic et al., 2010) and batteries (Key et al., 2011; Hung et al., 2012). These studies particularly benefit from the highest avail- able magnetic fields, due to the importance of elements such as lithium that possess large electric quadrupole moments. Future Prospects and International Perspective on NMR There is no doubt that the importance of NMR measurements will continue to expand into new scientific areas as new variants of these measurements are invented and as higher fields lead to further improvements in resolution and sensitivity. Since the discovery of NMR (resulting in Nobel prizes to the American physicists I.I. Rabi, in 1944, and E.M. Purcell and F. Bloch, in 1952), the United States has played a leading role in the development of NMR spectroscopy. Many of the critical developments in multidimensional NMR, in solid-state NMR methods and their underlying theory, in DNP technology, and in the exploration of applications in chemistry, biochemistry, biology took place in this country. (MRI and functional MRI were also first proposed and demonstrated here.) However, there is a consen- sus in the NMR community that the U.S. leadership role has eroded over the past 10 years. This is certainly true in the area of high-field NMR magnets. When 900 MHz (21.1 T) NMR magnets became available around 2002, approximately 15 were installed in the United States, 10 of which were purchased with federal government funds (NIH or DOE, plus the wide-bore 900 MHz magnet constructed at NHMFL). Relatively few NMR magnets above 800 MHz (18.8 T) were installed here in subse- quent years. Meanwhile, magnet technology has advanced to the point where a 1.0 GHz (23.5 T) NMR magnet was installed at the European Center for High Field NMR in Lyon, France, in 2010. Plans exist to install at least one 1.2 GHz (28.2 T) NMR magnet in Europe, at a new NMR center in the Netherlands. Additional 1.2 GHz NMR magnets are under negotiation for other European sites. Two 950 MHz NMR magnets were installed recently in this country, one with federal funding (NIH), the other purchased entirely by private funds. Each increment in magnetic field strength produces an improvement in NMR data, through increased resolution and sensitivity, as explained above. Magnetic field strength is not the only significant parameter in an NMR-based research project. Innovations in ancillary technology and RF pulse sequence methods, new approaches to data analysis, improvements in sample quality, and clever choices of scientific problems are also highly significant. For these reasons, U.S. NMR research groups that do not have access to the highest available fields can continue to make important scientific contributions. However, if the country were to fall further behind in NMR magnet technology, the most interesting and important

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High Magnetic Fields in Chemistry, Biochemistry, and Biology 75 problems, involving systems with the greatest complexity, biological relevance, and technological impact, would be solved elsewhere. It would also become increasingly difficult for research groups here to attract the brightest and most productive Ph.D. students and postdoctoral fellows, as it is natural for young scientists to prefer better-equipped research labs for their training. Investment in high-field NMR magnet technologies is highly leveraged. While this discussion is focused mainly on the importance of a small number of cutting- edge NMR magnets and spectrometers, it can be expected that demonstration of the scientific impact of these instruments will ultimately lead to the production of larger numbers of similar instruments, in more cost-effective ways and with enhanced technologies, to enable basic research in the chemical and biological sciences, in academic or national laboratories as well as the commercial sectors. Thus, the eventual impact of the initial cutting-edge instruments will expand beyond the results of specific experiments performed with these initial instru- ments. In addition, as magnet technology improves to meet the challenges of the next generation of NMR magnets, the cost of moderately high-field instruments, which are more widely distributed among individual research labs and institutions, is likely to decrease. The cost of a 1.2 GHz NMR magnet is approximately $20 million. To satisfy the likely demand for measurement time on a 1.2 GHz NMR system in the United States, at least three such systems would need to be installed by early 2015. More- over, planning for the next-generation instruments, likely a 1.5- or 1.6-GHz class system, should be under way now to allow for steady progress in instrument devel- opment. Given the size of the NMR community in the United States (more than 100 active research groups), the advantages of high-field NMR data discussed above, and the fact that each NMR data set requires hours to days of measurement time, the committee expects that three 1.2 GHz NMR systems would easily be used to full capacity. There is currently no mechanism by which funds on this scale can be obtained through the conventional peer-review processes at NIH or NSF or DOE. While the United States has historically held a leadership position not only in the applications of NMR in physics, chemistry, and biology but also in the development of NMR instrumentation and methodology, this privileged position is vulnerable. For this country to remain at the forefront of NMR-based research, new funding mechanisms must be developed. FT-ICR Mass Spectrometry FT-ICR is a technique in which the mass-to-charge ratios of charged particles, especially molecular ions, are measured from the frequencies of their cyclotron motions in a strong magnetic field. Compared with other forms of mass spec- trometry, FT-ICR has the highest demonstrated precision and resolution, making

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76 High Magnetic Field Science and I t s A pp l i c a t i o n in the US it the method of choice for complex mixtures of molecules and for distinguishing among chemical species with nominally identical masses but different elemental compositions. Magnets for FT-ICR have stability, homogeneity, and bore diameter requirements similar to those of NMR magnets, so that advances in NMR magnet technology have a direct impact on FT-ICR instrumentation. The resolving power (defined as M/DM, where M is the molecular mass and DM is the minimum mass difference that produces separate peaks in the mass spectrum) of an FT-ICR instru- ment increases linearly with increasing field; the accuracy of mass determinations and upper mass limit increases quadratically with field (Marshall and Guan, 1996). Thus, as with NMR, higher-field FT-ICR instruments have broader applications, yield data that are more informative, and permit experiments on systems with increasing complexity. Currently, the highest-field FT-ICR systems operate at 15 T and are located in several labs in the United States, Asia, and Europe. A 21 T system is under construction, through a joint effort involving NHMFL and Bruker Daltonics. The 21 T FT-ICR system will be made available to outside users as an NHMFL facility when completed. In chemical applications, FT-ICR allows the chemical formulae of individual species to be determined in complex mixtures, such as mixtures produced by com- binatorial chemistry, polydisperse synthetic polymers, and extracts from soil and plant matter. The FT-ICR group at NHMFL has pioneered the application of FT- ICR in petroleum chemistry, motivated by the fact that naturally occurring crude oil contains many thousands of chemical components, with distributions that vary with geographical location. Resolution and identification of these components requires the very high resolving power of a high-field FT-ICR instrument (M/DM > 105 for a mass-to-charge ratio of 1,000 at 15 T). NHMFL scientists have recently used FT-ICR to analyze petroleum samples from the Gulf of Mexico, following the Macondo oil spill, in order to track the chemical transformations that this mate- rial undergoes through evaporation, microbial degradation, and photochemical degradation, at various locations (Rodgers, 2011). In biological applications, FT-ICR mass spectrometry has emerged as a tool with immense impact in metabolomics (see discussion of NMR in metabolomics above) and proteomics, the study of protein structure and function via highly parallel large-scale data collection. ICR has particular advantages in the tandem Mass Spec-Mass Spec experiments, where proteins are identified not only by their total molecular weight but also by their patterns of fragmentation. FT-ICR is a forefront method because of the accuracy of the derived mass-to-charge ratios, and the measurement precision is currently limited by the availability of high magnetic field instrumentation (among other instrument issues). Development of higher magnetic field instruments is expected to increase the complexity of molecules amenable to analysis, and therefore more impact in human biology can be expected. It is furthermore expected that if instruments above 21 T can be implemented,

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High Magnetic Fields in Chemistry, Biochemistry, and Biology 77 single molecule analysis may become possible. The implications of studies at the single molecule level are many. Such highly sensitive analytic tools can be expected to allow better studies of the many microbes, including pathogenic microbes, that have not so far been successfully cultured in laboratories. Electron Paramagnetic Resonance EPR shares many of its basic principles with NMR, except that electron (rather than nuclear) spins are observed. Since the magnetic moments of electron spins (at g = 2) are 660 times larger than those of nuclear spins, EPR frequencies in chemical and biological applications are typically in the 9-400 GHz microwave range, with magnetic fields of 0.3-14 T. EPR at higher fields depends on somewhat exotic terahertz radiation sources but has been achieved in certain cases. Currently, high-field EPR is limited primarily by the properties and expense of the radiation sources, not by the properties of available magnets, so that EPR is not a major driver for magnet development. This situation could certainly change in the future. Nonetheless, high-field EPR is a growing field with important applications in chem- istry and biology, as higher fields produce greater spectral resolution and provide sensitivity to molecular motions on a wider variety of timescales. In particular, in structural biology, measurements of magnetic dipole-dipole couplings between electron spins, using pulsed EPR techniques, have become increasingly common as a means of determining distances in the 10-100 Å range between electron spin labels in proteins and nucleic acids. Such measurements are complementary to the shorter-range distance information available from NMR. Challenges in biochemi- cal preparative methods, magnet development, and microwave instrumentation are all important in the future of this field, and the development of higher field magnets for new high-field EPR spectrometers will be important for chemistry and structural biology over the next decade. Conclusion and Recommendation Conclusion: Nuclear magnetic resonance (NMR) spectroscopy is one of the most important and widely used techniques for structural, dynamical, and mechanistic studies in the chemical and biological sciences. However, in recent years, U.S. labs have failed to keep up with advances in commercial NMR mag- net technology. If this trend continues, the United States will probably lose its leadership role, as scientific problems of greater complexity and impact are solved elsewhere. NMR methods continue to improve, and new applications for NMR continue to be discovered and explored, driven in large part by the ongoing development of

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78 High Magnetic Field Science and I t s A pp l i c a t i o n in the US higher field superconducting magnets with high stability and homogeneity. Since the discovery of NMR, the United States has played a leading role in all aspects of NMR spectroscopy. However, the highest-field NMR magnets in this country are limited to the 900-950 MHz range (21.1-22.3 T), while a 1.0 GHz NMR system (23.5 T) was installed in Europe in 2010 and 1.2 GHz NMR systems (28.2 T) have been ordered by several European labs. The cost of a 1.2 GHz NMR magnet is approximately $20 million. There is currently no mechanism by which funds on this scale can be obtained through the conventional peer-review processes at the National Institutes of Health or the National Science Foundation or the Department of Energy. It is clear that new mechanisms must be devised in order to fund and site high- field NMR systems in the United States. Recommendation: New mechanisms should be devised for funding and sit- ing high-field NMR systems in the United States. To satisfy the likely demand for measurement time in a 1.2 GHz system, at least three such systems should be installed over a 2-year period. These instruments should be located at geo- graphically separated sites, determined through careful consultation with the scientific community based on the estimated costs and the anticipated total and regional demand for such instruments, among other factors, and managed in a manner that maximizes their utility for the broad community. Moreover, planning for the next-generation instruments, likely a 1.5 or 1.6 GHz class system, should be under way now to allow for steady progress in instrument development. References Bhate M.P., B.J. Wylie, L. Tian, and A.E. McDermott. 2010. Conformational dynamics in the selec- tivity filter of KcsA in response to potassium ion concentration. Journal of Molecular Biology 401:155. Buannic, L., F. Blanc, I. Hung, Z. Gan, and C.P. Grey. 2010. Probing the local structures and protonic conduction pathways in scandium substituted BaZrO3 by multinuclear solid-state NMR spec- troscopy. Journal of Materials Chemistry 20:6322. Cady, S.D., K. Schmidt-Rohr, J. Wang, C.S. Soto, W.F. DeGrado, and M. Hong. 2010. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463:689. Fu R., X. Wang, C. Li, A.N. Santiago-Miranda, G.J. Pielak, and F. Tian. 2011. In situ structural char- acterization of a recombinant protein in native Escherichia coli membranes with solid-state magic-angle-spinning NMR. Journal of the American Chemical Society 133:12370. Hung, I., L. Zhou, F. Pourpoint, C.P. Grey, and Z. Gan. 2012. Isotropic high field NMR spectra of Li-ion battery materials with anisotropy >1 MHz. Journal of the American Chemical Society 134:1898.

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