Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) is a second method adapted from physics that is now commonly used to reveal the structures of proteins and other molecules. It does not require proteins in crystal form, which means that it overcomes some of the problems faced by crystallographers.

The First Synchrotron Experiments

Ken Holmes was a Ph.D. student in Rosalind Franklin's laboratory in London from 1955 to 1959. After spending two years as a postdoctoral student at the Children's Hospital Boston, he returned to the Laboratory of Molecular Biology (famous for its scientists having accrued 13 Nobel Prizes) in Cambridge, England. In 1968 he became a director at the Max Planck Institute for Medical Research (recipient of four Nobel Prizes) in Heidelberg,

Germany. In the late 1960s Holmes and his colleague Gerd Rosenbaum carried out the first experiments on protein structures using X-rays produced by the electron synchrotron DESY, a huge ring-shaped particle accelerator used in physics experiments. Today electron synchrotrons or storage rings have become the main source of X-rays for diffraction experiments to obtain the structures of proteins and other molecules. In the following personal interview with the author, he shares some memories of those pioneering experiments.

Ken Holmes is former head of the Max Planck Institute for Medical Research in Heidelberg, Germany, and a pioneer in the use of synchrotron radiation for the study of biological molecules. In the 1950s he was a doctoral student under Rosalind Franklin. (Russ Hodge)

While I was in Cambridge, I became interested in trying to get better sources of X-rays. At the time the method people were using was essentially the same as that discovered by [Wilhelm] Röntgen, which involved creating a beam of electrons at moderately high energy [in a vacuum] and shooting them at a metal, in our case a chunk of copper, known as the anode. Why does this produce X-rays? The beam knocks electrons out of their deep shell in the copper atoms and as they return to that shell they give off fluorescent energy in the form of X-rays. However, the electron beam carries quite a bit of energy and so the beam easily burns a hole in the metal, or melts it. To stop this [from] happening you have to cool the metal, and rotate it [the anodes are often rotating metal discs]. Cambridge had a very good workshop that was making rotating anode X-ray tubes [called "tubes" because Röntgen's original apparatus was in a long glass tube], but their source of electrons—a sort of gun that shoots them at the metal—was not much good. So with my colleague Bill Longley we developed a much better electron gun and connected it with their anode. The head of the workshop had absolutely no faith in the whole thing and only grudgingly gave us one of their beautiful anodes. Nevertheless, the new X-ray tube worked, and we successfully marketed it. The intensity of the X-rays was much better but still far short of what we wanted. To get more intensity you need more electrons, but this produces more heat. To dissipate the heat you spin the disc faster. However, you can only spin a disk of metal

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so fast before it flies apart. Moreover, you've got to cool the system somehow. One quickly reaches physical limits. Therefore, I began looking around for even better sources of X-rays.

At that stage the German government and University of Hamburg were planning to build a very powerful electron accelerator facility called the Deutsches Electronen Synchrotron (DESY). This is a huge ring-shaped tunnel containing an evacuated stainless steel tube in which electrons could be accelerated. This also begins with an electron gun, but instead of firing a beam of electrons into metal, it fires electrons into the ring. They are already traveling at about the speed of light, but then they are accelerated even more, and as that happens, they get heavier. When they reach their maximum energy they are deflected into a target, where they produce a menagerie of subatomic particles. Electromagnets mounted along the tunnel keep the electrons moving in a circle, rather than a straight line, but curving their trajectory causes the release of energy—intense radiation known as synchrotron radiation covering all wavelengths from infrared to X-rays—that fly off at a tangent at every bending magnet. This is a curse for the high-energy physicists (it increases their electricity bill enormously) but a blessing for us. If you insert a suitable window you can let the radiation out of the ring. One can then select out the X-rays with a suitable crystal monochroma-tor. It turns out, you end up with an intense X-ray source. The X-rays then need to be focused into a tiny beam and directed at the biological sample, often a crystal, but our work involved viruses and muscle fibers.

In 1963 I wrote to DESY and suggested that we ought to use the ring as an X-ray source. I got an encouraging response, but at the time I was still in Cambridge, England, and it was not easy to do a project across international borders. Then I received the appointment in Heidelberg. One of my first students was a physicist named Gerd Rosenbaum, who had just finished his undergraduate studies at DESY. He had done experiments there and knew the whole set-up. He went back to Hamburg and began setting up our equipment. When the time came for the experiments, we were under intense time pressure. We only had 16 hours to mount our equipment, take measurements, and put everything back. We had to use an existing lab—physicists at the synchrotron had already set up an experimental laboratory to utilize the synchrotron radiation, but their interest was in ultraviolet radiation, not X-rays, so their setup was quite different from what we needed. They warned us: "You cannot perturb anything here; if you move anything, you have to put it back just the way it was." Somehow we managed, and in those 16 hours we got the first X-ray diffraction patterns ever taken with synchrotron radiation, actually with a bit of muscle fiber.

Even the synchrotron was not an ideal source of X-rays. The electromagnets in the ring are part of tuned oscillating circuits; in essence they are big inductive coils with condensers attached to them, and their magnetic field goes up and down 50 times a second in a synchronized way to match the ever increasing mass of the circulating electrons [hence the name synchrotron]. What this means for our experiments is that the electron beam in the ring is produced and killed 50 times a second, which is not

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highly efficient! And it only gives off X-rays when it is at the peak of its energy, which is only 10 percent of the total time it operates. But DESY was already planning to create a "storage ring," where a steady beam of electrons at one energy would be kept in a constant orbit. If we could build an experimental station on that ring, we would have a much better continuous source of X-rays that could be used on a permanent basis for biological experiments. We built that station as a service facility of the European Molecular Biology Laboratory; over the last 30 years it has been used to obtain structures by protein crystallography of thousands of biological macromolecules by scientists from all over Europe.

Synchrotron radiation has become very important. Nowadays there are a couple of dozen electron storage rings distributed round the world that have been custom built as intense X-ray sources. Although the source is nowadays a storage ring rather than a synchrotron, the radiation is still known as synchrotron radiation. These sources have yielded data for the solution of tens of thousands of atomic structures of biological macromol-ecules, some, such as the ribosome, of immense size and complexity. The growth of protein structure determination by X-ray diffraction from an important but esoteric method to one of the pillars of modern molecular biology would not have been possible without synchrotron radiation.

Note: In a second interview in chapter 2, Holmes recounts some of his memories of working with Franklin in Cambridge in the late 1950s.

To build a crystal, proteins have to be captured in a liquid at high concentrations and then slowly dried until they lock onto each other in symmetrical arrangements. This method cannot be used to study all protein structures or to answer all of scientists' questions about them. First, not all proteins form crystals of a high enough quality to be studied. Second, crystals are often highly artificial forms of molecules. A protein's natural environment is liquid, where it moves in a flexible way as it finds and binds to partners. That flexibility is mostly lost in the rigid structure of a crystal. NMR avoids some of these problems because it can look at proteins in liquids or even a solid form.

The principles behind NMR were discovered in the 1930s by Isidor Rabi (1898-1998), a physicist at Columbia University. He was using molecular beams to investigate the forces that hold electrons to the nuclei of atoms, work that earned him the 1944 Nobel Prize in physics. It also had practical applications in the development of radar, a project that Rabi was recruited to work on in World War II. Two physicists who had been likewise recruited, Felix Bloch (1905-83) and Edward Purcell (1912-97), adapted NMR so that it could be used to investigate liquids and solid objects. Their work led to the 1952 Nobel Prize in physics. NMR slowly became an important method for the determination of structures of biological molecules thanks to the efforts of Kurt Wuthrich (1938- ), a Swiss chemist who now heads laboratories at the Swiss Federal Institute of Technology in Zurich and the Scripps Institute in La Jolla, California. Wuthrich began working with NMR when he joined Bell Laboratories in Murray Hill, New Jersey, in 1967, where he had access to one of the best instruments in existence at that time. He used it to study the structure and behavior of proteins in liquids; thanks largely to his efforts, it has become a standard tool in structural biology, drug discovery, and related fields. His accomplishments were recognized with the award of the 2002 Nobel Prize in chemistry.

Obtaining a molecular structure requires finding positions of single atoms and establishing their positions relative to other atoms in the same molecule. NMR works by both identifying atoms and "interrogating" them about others that are nearby. It applies a very strong magnetic field to a liquid containing the protein (and sometimes other types of molecules) that a scientist wants to study. When placed in a magnetic field of a precise strength, the nuclei of some atoms absorb an amount of energy that can be precisely measured. Different strengths of fields are required to make different types of atoms behave this way, so energy measurements can reveal the presence of specific atoms. Finding their locations relative to each other requires relaxing the magnetic field again.

This effect is like moving a strong magnet next to a compass and then removing it. When it is close, the magnetic field draws the needle of the compass. Removing the magnet again makes the needle return to its normal position. The same effect happens with the nuclei of atoms in an NMR experiment. The magnetic field aligns them; when it is relaxed, they return to their normal state, but the way that they do so depends on what other atoms are nearby. Thus, the properties of one atom produce a "signature" that reveals what other atoms are nearby, and this can be used to reconstruct a map of a protein or another molecule.

NMR can only provide information about small regions of molecules. This limitation means that it usually does not provide the structure of an entire molecule. On the other hand, it can examine proteins in liquids, which is close to their natural state. This means that researchers can also observe changes in its structure, during processes such as the binding of a small molecule to a protein. That gives a look, for example, at how a drug interacts with its target.

The same principles and technology underlie magnetic resonance imaging (MRI), a medical technique. Instead of detecting mineralized substances such as bone, the way an X-ray machine functions, MRI detects the presence of particular fluids. This allows it to be used to study the structure and activity of the brain and other organs.

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