Structure of the ribosomal protein L9 as determined by NMR.

Structural Studies of Molecules

Imagine yourself trying to take a picture of a molecule so you can understand how it performs its function. The problem is that the molecule is far too small for you to be able to see it with any kind of optical lens. You need to rely on common properties of a large ensemble of identical molecules, and use these to infer the structure of the molecule. This is what X-ray crystallographers and NMR spectroscopists do.

Chemists and biochemists perform experiments to investigate chemical phenomena. They are usually very good at producing models of what happens, but in many cases, the complexity of the reactions or the size of the molecules is too great to be able to create a unique model that fits all the chemical data available. This is especially true in the field of biochemistry. During the 1940s and 1950s, two very powerful methods emerged that today enable biochemists to elucidate the structures of large proteins and nucleic acids, as well as organic and inorganic compounds. This branch of science that deals with the elucidation of the structures of biomolecules as related to biochemistry is known as structural biology. The two most useful methods structural biologists have available are X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. With these very powerful techniques at their disposal to make accurate models of the three-dimensional structures of biomolecules such as protein enzymes and nucleic acids, the structural subtlety of many of the chemical reactions that take place in living cells can be elucidated. The structures of biomolecules that lend themselves to the highly ordered self-aggregation known as crystallization can be determined by X-ray crystallography. Much information is available in the literature about this method. The scope of this article, though, will be limited to discussion of the uses of NMR spectroscopy in biochemical research.

Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is a very powerful method that relies on the interaction of certain atomic nuclei with radio waves and strong magnetic fields. Equipped with a powerful, superconducting magnet (typical field strengths are around 10-20 Tesla), a radio-frequency probe, and a large creative set of NMR pulse sequences, structural biologists are able to investigate the structures of biomolecules in solution. With three-dimensional atomic structural information available, biologists can get clues about how these molecules function within the organism in which they are found.

How NMR Works

The way NMR works can be explained to some extent by an analogy to semi-classical mechanics, as follows: Certain atomic nuclei, such as protons, have an atomic spin of 1/2. When these protons are exposed to a homogeneous atomic field, they begin to rotate about an axis and thus acquire rotational momentum. When they start rotating, they also begin to precess about their rotational axis. They reach equilibrium after some time. If radio-frequency (RF) pulses are directed at them, and the frequency is near the precession frequency (or the power of the RF pulse is high enough and the pulse is short), the nuclei absorb the energy until they are saturated. If the RF pulse is turned off and one listens with a radio receiver, one can detect the precession frequencies of the nuclei as their energy is released and they relax back to equilibrium. In molecules with more than one proton, one can see several frequency lines, each corresponding to a nucleus in the molecule. The accompanying figure shows a one-dimensional (left) and two-dimensional (right) NMR spectrum of the ribosomal protein L9, which contains about 1,500 proton nuclei distributed throughout the structure. By assigning 95-100% of the nuclei to their respective frequency, and by finding the distances between them, the NMR spectroscopist can calculate the three-dimensional structure of the molecule (shown at top).

NMR researcher Jarle Lillemoen adding his L9 protein sample to the NMR machine.

With NMR, if one measures the rate of relaxation of the nuclei back to equilibrium, one can also get an understanding of what types of motions are present in the important biomolecules. A molecule that has been subjected to this type of study is ribosomal protein L9, a protein that serves an architectural role in the ribosome. L9 consists of two RNA-binding domains separated by a long nine-turn a-helix. Its structure was determined by X-ray crystallography and verified by NMR. By measuring backbone amide exchange rates and nitrogen-15 relaxation rates, it was found that the protein has a very stable central helix that keeps the two RNA-binding domains separated, and that the RNA-binding regions are more flexible than the rest of the molecule. This information can be used to develop a coherent model of how the ribosome is able to perform its function, translation of RNA into proteins.

To summarize, a lot of chemical and biochemical investigations can be performed without structural information, but with structural and dynamic information available, one can make more complete models of how enzymes or large complexes such as the ribosomes perform their functions in the living organism.

Biochemistry NMR Web Site Links

Dr. Arthur G. Palmer III

Dr. Lewis E. Kay

Dr. Kurt Wüthrich

CrystaLinks - Crystallography resource Web site.

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