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The World's Most Powerful NMR Magnet Arrives at TSRIThe largest nuclear magnetic resonance (NMR) magnet ever constructed arrived last week at The Scripps Research Institute (TSRI), where it was assembled into the most sensitive NMR instrument on the planet. The new NMR, referred to by its maximum radio frequency, 900 MHz, is now the centerpiece of TSRI's premier collection of NMR instruments. "It's fantastic," says Peter Wright, professor and chair of the Department of Molecular Biology. "The capabilities of this instrument take us to a new level." Now in its permanent home in one corner of the Buddy Taub Center for Molecular Structure and Design, the 900 MHz instrument sits next to the 800 MHz instrument that has been in the same building since 1998. Although appearing only a little taller and a little fatter than the 800, the 900 represents a major breakthrough in technology. "It's rather a technological triumph," says Associate Professor Jane Dyson of the new machine. The instrument, which is the first of its kind and has been several years in the making by its German manufacturer, Bruker Instruments, Inc., is currently being cooled down to its operating temperature of 2 degrees Kelvin above absolute zero with 5000 liters of nitrogen and up to 4,500 liters of liquid helium. The instrument's magnet is actually several miles of ultra-high technology Niobium-Tin and Niobium-Titanium superconducting coils sitting directly in the helium bath, which is surrounded by a vacuum dewar and a liquid nitrogen bath. This magnet will be charged with over two hundred amperes of persistent super-conducting current. Through induction, this current will produce a stable magnetic field of 21 tesla, which is very large compared to a hospital MRI 2 tesla magnet. In addition, the field is remarkably homogenous: better than one part per billion over a couple of cubic centimeters volume. Once filled and charged, the instrument will be calibrated, tested for a few weeks, then put to use by research groups at TSRI. Improvements to Resolution and Sensitivity"There are already problems waiting to go into it," says Wright. "Instantly—as soon as it's available." Some of those problems will take advantage of the improvements of the 900 over existing lower field instruments at TSRI. For instance, the 900 is expected to be 40 to 50 percent more sensitive (peak heights) and have greater resolving power (peak separations) than the 800 MHz instrument. The increased resolution will help separate peaks of two close chemical shifts that would normally overlap. "The first thing we do will probably be to exploit this resolution to study systems that were not possible before," says Dyson. The increased sensitivity should allow measurements to be taken on samples with lower protein concentrations. Typically, some proteins, big and small, don't behave well at high concentrations. Modern experiments require the protein to be stable around 100 µM to1mM, for instance, but this is impossible for some systems because the molecules may dimerize, aggregate, or precipitate at that concentration. The concentration can be reduced but only at the cost of reducing the signal-to-noise ratio, with the risk of losing the signal altogether. But the increased sensitivity of the 900 should allow concentrations to be lowered even further before the signal disappears. However, even with the increased resolution and sensitivity, many interesting systems remain elusive to NMR because they are either too big, too insoluble, or too complicated. The size limitations for structures that may be obtained through conventional NMR, for instance, is around 30 to 40 kD, but many proteins are simply much larger than this. Membrane proteins are also complicated to deal with. These proteins, which make up about one third of all the genes in the human genome, are notoriously hard to crystallize and would make excellent problems for NMR spectroscopists if they weren't so large and insoluble. Then there are partially folded and unfolded systems that are too complicated to study with conventional NMR because the increased flexibility of the unfolded polypeptides significantly increases the difficulty of analyzing the spectra. Much can be gained by studying the partly folded states of proteins and observing how they fold. Do they make secondary or tertiary structures? How flexible are their backbones and how flexible are their side chains? "All these things we can get at," she says, "but because the spectra are so overlapped, we need this big machine." "Until now we haven't had the tools to study these things—the unfolded proteins, the membrane proteins, the huge proteins—you can't look at them because they are so difficult," says Dyson.
The Huge Advantage of TROSYA major breakthrough has come in the form of a new technique that is sure to take advantage of the size of the new 900 MHz instrument. This technique is called transverse relaxation optimized spectroscopy (TROSY). TROSY is a technique that suppresses the transverse nuclear spin relaxation—a quantity that causes the deterioration of NMR spectra for larger molecular structures. "What TROSY lets you do is work on really big systems," says Molecular Biology Professor Jamie Williamson. Using TROSY, the upper size limit for molecular structures that can be studied with NMR can be extended to include those that are hundreds of kD in size. "This is one of the things we are going to do with the 900," says John Chung, director of TSRI's Biomolecular NMR facility. The optimal frequency for the TROSY effect is calculated to be near one GHz, and since 900 MHz is closer to this than any instrument ever before, Wright, Dyson, Williamson and their colleagues believe that they will be able to effectively apply TROSY to very high molecular weight systems on the new instrument. "Up to this point we have been limited to about 30 kD," says Williamson, whose research involves looking at protein/RNA complexes such as Rev and Tat protein bound to HIV RNA. Another set of structures he studies are intermediates in the assembly of the bacterial ribosome, which when fully assembled is about 2.7 million daltons. "Now we can ask how do these pieces that we look at look in the context of the larger assembly," he says. Moreover, Kurt Wüthrich, who was the first to recognize and exploit TROSY at ultra-high magnetic field strengths with his colleagues in Switzerland, is coming to TSRI this year as a visiting professor, while maintaining his position at ETH in Zurich. "This is an NMR Mecca right now—this is where it's all happening," says Wright. "And now, with Wüthrich coming, it's going to be even more of a Mecca." NMR—An Old Friend to Modern BiologyDiscovered in 1946 by independent groups at Stanford and Harvard Universities, NMR refers to the ability of atomic nuclei to reorient themselves in a magnetic field when exposed to radiation of a particular "resonant" frequency in the radio band. Atomic nuclei contain charged particles with spin, which according to Maxwell's equations, induces a magnetic field. Though small, the magnetic "moments" of these nuclei makes them sensitive to an external magnetic field. In an NMR magnet, the nuclei act like tiny bar magnets and tend to align themselves preferentially in a particular configuration, while also undergoing spinning motions similar to the gyroscopic precessions of bicycle wheels or spinning tops under an external torque. Any fluctuating magnetic field orthogonal to that of the NMR magnet will perturb the alignment of the nuclear magnetic moments away from the equilibrium configuration, but only if the frequency of the fluctuating field is precisely equal to the precession frequencies of the nuclear magnetic moments. These are called the resonant, or Larmor, frequencies and are proportional to the field strength of the NMR magnet. TSRI's new 21 tesla magnet, for instance, causes protons to precess at precisely 900 MHz. Movement of atomic nuclei in the NMR as they go in and out of resonance causes small but measurable induced voltages, and it is this signal which is being measure in the NMR experiment. An NMR spectrometer will scan a broad range of radio frequencies and record all the resonances as a spectrum. Atoms like 1H, 13C, or 15N, have nuclear spin and give rise to a sharp NMR signal, whereas atoms like 12C, and 16O have no nuclear spin and therefore no signal. This makes the technique of NMR very powerful because different spectra can be taken with molecules that have been selectively isotopically labeled with atoms that have or do not have spin. In an NMR experiment, a sample in a long tube is inserted into the magnet, and the resonant responses of the atoms in the sample over a range of frequencies is recorded. These responses are influenced by the shape of the molecule in which the atoms reside—by their proximity to other atoms in the molecule. An NMR spectrum is unique for a particular molecule, and the structure of a molecule can be determined from its spectrum. NMR is more than simply a useful tool for chemists and biologists. It is ubiquitous at universities, biotech companies, and biomedical research institutes. TSRI is a leader in high-powered NMR instrumentation, with over 13 instruments above 500 MHz. The 900 MHz spectrometer is the latest of a large number of instruments, but there is no doubt that, for a time, it will be the greatest—here or anywhere. "The fact that [the 900] is coming to TSRI is a big deal," says Wright, "because it allows us to push the frontiers of NMR research on biomolecules." |
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