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Sci Tech
Knowing how proteins function in cells
Fenn, of the Virginia Commonwealth University in Richmond, VA, USA, transformed proteins into gas phase by spraying the sample using a strong electrical field.
THE NOBEL Prize in Chemistry for 2002 is being shared between scientists in two important fields: mass spectrometry (MS) and nuclear magnetic resonance (NMR). The Laureates, John B. Fenn and Koichi Tanaka (for MS) and Kurt Wüthrich (for NMR), have contributed in different ways to the further development of these methods to embrace biological macromolecules. Chemists can now rapidly and reliably identify what proteins a sample contains. They can also produce 3D images of protein molecules in solution. Events in the cells are controlled by nucleic acids (such as DNA) that may be termed the cells' "directors", while the various proteins are the cells' leading actors. Protein research itself is not new, but proteomics, i.e. studies of how different proteins and other substances act together in the cell, is a relatively new field of research. As the gene sequences of more and more organisms have been mapped and the research frontier has advanced, new questions have cropped up: how can it be that man's 30,000-or-so genes code for hundreds of thousands of different proteins?
Mass spectrometry now allows us to identify a substance in a sample, rapidly, on the basis of its mass. This technique has long been used by chemists on small and medium-sized molecules. The method is so sensitive that it is possible to trace very small quantities of each type of molecule. Doping and drug tests, foodstuff control and environmental analysis are examples of areas where mass spectrometry is now in routine use. Macromolecules may be large in comparison with other molecules but we are nevertheless dealing here with incredibly small structures weighing 10{+-}{+1}{+9} g. How to weigh something that is so small? The trick is to cause the individual protein molecules to let go of each other and spread out as a cloud of freely hovering, electrically charged protein ions. A common method of subsequently measuring the mass of these ions and hence identifying the proteins is to accelerate them in a vacuum chamber where their time of flight (TOF) is measured. They "reach their targets" in an order determined partly by their charge and partly by their mass. The fastest ones are those that are lightest and have the highest charge.
Today there are two principles for causing proteins to transform into the gas phase without losing their structure and form, and it is the discoverers behind these methods that are being rewarded jointly with half the Nobel Prize in Chemistry. In one of these methods, of which John B. Fenn is the originator, the sample is sprayed using a strong electrical field to produce small, charged, freely hovering ions.
The other method, instead, uses an intense laser pulse. If this is done under suitable conditions (as to the energy, structure and chemical environment of the sample) the test molecules take up some of the energy of the laser pulse and become released as free ions. The first person to show that this phenomenon, soft laser desorption, could be used for large molecules such as proteins was Koichi Tanaka.
Kurt developed an idea about how NMR could be extended to cover biological molecules such as proteins.
Fenn's contribution
During 1988 John B. Fenn published two articles that were to mean a breakthrough for mass spectrometry with "electrospray" for macromolecules. In the first, studies of polyethylene glycol molecules of unknown mass showed that the method could handle large molecule masses with high charges. The second publication reported the use of the method on medium-sized whole proteins as well. The release of ions is achieved by spraying the sample using an electrical field so that charged droplets are formed.
As the water gradually evaporates from these droplets, freely hovering "stark naked" protein molecules remain. The method came to be called electrospray ionisation, ESI. As the molecules take on strong positive charges, the mass/charge ratio becomes small enough to allow the substances to be analysed in ordinary mass spectrometers. Another advantage is that the same molecule causes a series of peaks, since each can take up a varying number of charges. While this complicates the pattern, at first confusing the researchers, it also gives information that makes identification easier.
Tanaka's contribution
At the same time exciting things were happening in another part of the world. At the Japanese Shimadzu instrument company in Kyoto, a young Japanese engineer, Koichi Tanaka, reported an entirely different technique for the first critical stage. A year later Tanaka showed that the protein molecules could be ionised using soft laser desorption (SLD). A laser pulse strikes the sample which, unlike in the spray method, is in a solid or viscous phase. When the sample takes up the energy from the laser pulse it is "blasted" into small bits. The molecules let go of one another, released as intact hovering molecule ions with low charge, which are then accelerated by an electrical field and detected as described above by recording their time of flight. Tanaka was the first to demonstrate the applicability of laser technology to biological macromolecules. The principle is fundamental for many of today's powerful laser desorption methods. .
NMR for biological
macromolecules
Where mass spectrometry gives answers to questions about e.g. a protein, such as "what?" and "how much?". NMR in one sense answers the question "what does it look like?" Even the largest proteins are too small to be studied at sufficient resolution with any type of microscope. To be able to form a picture of what a protein really looks like, then, other methods must be used. NMR (Nuclear Magnetic Resonance) is one such method. By interpreting the peaks in an NMR spectrum one can draw a three-dimensional picture of the molecule being studied. One finesse is that the sample can be in a solution, in the case of proteins their natural environment in the cell.
The applicability of the NMR method was initially limited by its low sensitivity: it required incredibly concentrated solutions. . By interpreting the signals in an NMR spectrum it was thus possible to gain an idea of the appearance of the molecule, its structure. The method was successful for relatively small molecules but, for larger ones, it was hard to differentiate between the resonances of the different atom nuclei. An NMR spectrum of this kind could look like a grass lawn in section thousands of peaks where it was impossible to decide which peak belonged to which atom. The scientist who finally solved this problem was the Swiss chemist Kurt Wüthrich.
Kurt Wüthrich showed that NMR was possible for proteins. At the beginning of the 1980s, Kurt Wüthrich developed an idea about how NMR could be extended to cover biological molecules such as proteins. He invented a systematic method of pairing each NMR signal with the right hydrogen nucleus (proton) in the macromolecule. The method is called sequential assignment and is today a cornerstone of all NMR structural investigations. He also showed how it was subsequently possible to determine pairwise distances between a large number of hydrogen nuclei and use this information with a mathematical method based on distance-geometry to calculate a three-dimensional structure for the molecule.
The first complete determination of a protein structure with Wüthrich's method came in 1985. At present 15-20% of all the thousands of known protein structures have been determined with NMR. The structures of the others have been determined chiefly with X-ray crystallography; a few with other methods such as electron diffraction or neutron diffraction.
Tanaka, of the Shimadzu Corp. in Kyoto, Japan, used intense laser pulse to convert large molecules to free ions.
Areas of application for NMR with macromolecules
In many respects, the NMR method complements X-ray crystallography for structural determination. If the same protein is investigated with both methods, in the one case in solution and in the other crystallised, the same result is generally obtained, with the exception of certain superficial areas that are affected by the environment in both cases in the crystals by the tightly packed protein molecules, in solution by the surrounding molecules of the solvent. While the strength of X-ray crystallography lies in being able to determine accurately really large three-dimensional structures, the NMR method has other unique advantages.
The fact that the investigation takes place in a solution means that physiological conditions can be approximated. A particular strength of NMR is its ability to demonstrate unstructured and very mobile parts of a molecule. It is possible to elucidate the mobility, the dynamics, and how it varies along a protein chain. Isotope labelling can also be used to facilitate the identification of the atoms.
One example of NMR-determined protein structures comes from studies of the prion proteins involved in the development of a number of dangerous diseases such as mad cow disease.
NMR is also used in the pharmaceuticals industry to determine the structure, and hence the properties, of proteins and other macromolecules that can be interesting target molecules for new pharmaceuticals. Pharmaceutical molecules are designed to fit into the structure of the protein like a key in a lock.
The perhaps most important industrial use of NMR is in the search for small potential pharmaceutical molecules that can interact with a given biological macromolecule. If the small molecule binds to the large one, the NMR spectrum of the large molecule is normally changed. This may be used to "screen" a large number of pharmaceuticals candidates at an early stage of the development of a new drug.
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