1991 Nobel Prize Winners Sparked Fundamental Advances

The 1991 Nobel Prize winners in science were announced last month, and for the first time in 43 years, none of the laureates is from the United States. Yet their work--in nuclear magnetic resonance spectroscopy, phase transitions in materials science, and patch-clamp methodologies--collectively has influenced research in the U.S. and throughout the world. As is typical of Nobel Prize winners, their pioneering advances have changed the way science is done across the spectrum of scientific disciplines. Chemistry The Royal Swedish Academy of Sciences awarded the Nobel Prize in chemistry to Swiss chemist Richard R. Ernst, of the Eidgen”ssische Technische Hochschule (ETH) in Zürich, Switzerland. Ernst's contributions to the development of nuclear magnetic resonance (NMR) spectroscopy have made the technique nearly indispensable in many of today's scientific laboratories studying a wide range of phenomena, such as the three-dimensional structure of proteins, the chemical interactions of molecules in solution, and the effects of drugs on metabolic processes. Although Ernst has been active in the field of NMR spectroscopy for some 30 years, he was recognized by the Nobel committee primarily for two fundamental advances, Fourier-transform NMR (FT-NMR) and two-dimensional NMR (2D-NMR). NMR was developed in the 1940s by two independent groups, Felix Bloch's at Stanford University and Edward Purcell's at Harvard University; both men shared the Nobel Prize in physics in 1952. Although NMR's analytical potential was recognized early on, the technique's low sensitivity was a stumbling block to its widespread development. In NMR spectroscopy, the substance to be analyzed is placed in a magnetic field. Some of the atomic nuclei behave like tiny compass needles (called nuclear spins) and align themselves in the direction of the magnetic field. According to the laws of quantum mechanics, these nuclei are limited to certain discrete orientations, each of which is characterized by a different energy level. During the course of the analysis the nuclei are also exposed to a fluctuating radio signal. When the radio frequency exactly matches the resonance frequency of the nuclear spins, the nuclei jump between energy levels, inducing an electrical signal. The strength of this signal is then plotted as a function of frequency. In 1966, Ernst, then a research scientist in the analytical instrument division of Varian Associates in Palo Alto, Calif., and his supervisor, Weston A. Anderson, published a paper outlining a way to apply Fourier transformation--a powerful mathematical technique for filtering out background noise in instrumental analysis--to NMR (R.R. Ernst, W.A. Anderson, "Application of Fourier transform spectroscopy to magnetic resonance," Review of Scientific Instruments, 37:93-102). This advance paved the way for NMR's development as an essential and powerful analytical method. In FT-NMR, the slow radio-frequency sweep to which the sample is exposed is replaced by short, intense radio-wave pulses. The signals after each pulse are recorded, and a computer performs Fourier transform analysis on the data points. The resulting ten-fold--and sometimes hundred-fold--increase in sensitivity has made it possible to study very small quantities of material, as well as isotopes, such as 13C, that don't occur very often in nature. By the early 1970s, this breakthrough had been incorporated into commercially available NMR instruments. While the new technique's exquisite sensitivity was its most obvious advantage at first, Stanley Opella, professor of chemistry at the University of Pennsylvania, says that other benefits were far more important. "[Sensitivity] was really the least of it," he explains. "With pulsed radio frequencies, you have tremendous flexibility with what you do with that pulse." By adjusting their amplitude, phase, and timing, Opella says, one can use the pulses to "reveal all the underlying spin physics . . . and see their manifestations in the spectra." This means that researchers can study interactions between various substances--for example, the rates of chemical reactions or the dynamics of molecules in solution. In an essay written for the Philadelphia-based Institute for Scientific Information's Current Contents/Physical, Chemical, & Earth Sciences (23[27]:24, 1983), Ernst described the early unwieldiness of FT-NMR: "In 1964, we certainly did not foresee that this simple concept could revolutionize NMR. Initially, we did not have an online computer available. The free induction decays [NMR signals] were acquired in a time-averaging computer and punched on paper tape. The paper tape had to be carried from Palo Alto to IBM in San Jose to transfer the data to a bunch of cards. With the cards we went to the Palo Alto Computer Service Center, where the Fourier transformation and the plotting were done. "The entire process took about 48 hours in comparison with a normal spectrum being run in just 10 minutes! Nevertheless, we claimed in our paper a substantial time saving--and the readers believed it!" In 1976, Ernst, who had joined the ETH faculty in 1968, made another seminal contribution in this field when he developed two-dimensional NMR. This variation of FT-NMR vastly improved resolution over earlier methods and allowed NMR analysis of macromolecules. In 2D-NMR, the radio-frequency pulses used in FT-NMR are divided into two sequences, each one followed by a period with no pulses. The NMR signal is measured during the second pulseless period. This four-part pattern is repeated many times, but each time the duration of the first pulseless period--called the evolution period--is increased by constant increments. The resulting two-dimensional table plots NMR signal intensity in relation to both the point in time that each signal was measured and the length of the evolution period. The paper in which this breakthrough was published, "Two-dimensional spectroscopy. Application to nuclear magnetic resonance" (W.P. Aue, E. Bartholdi, and R.R. Ernst, Journal of Chemical Physics, 64:2229-46, 1976), has been cited more than 1,400 times through 1990, according to ISI's Science Citation Index (SCI). In 1987, the annual citation rate peaked at 177. And while citations have decreased somewhat since then, the 125-plus citations the paper received in 1990 indicate that it not only established the basis of a now-widespread technique, but also is still a springboard for more recent advances. The 1966 paper in which Ernst and Anderson introduced FT-NMR has a meager citation record in comparison to this latter one. Although the paper quickly became a classic, its 500 or so total citations do not come close to reflecting its fundamental importance to spectroscopy. This paper, like many others that came before and some that have appeared since, has become part of the uncited "body of scientific knowledge." Within a decade after it was published, nearly all NMR spectrometers used pulse excitation. "We all use everything that's in the first paper, but it's become so important that you don't even need to refer to it," says Opella. "It's like putting tires on a car. Everybody just assumes you have tires on your car. Now if you have square tires, that needs talking about." Since this work was published, Ernst and others have refined and expanded these methods. Using NMR, researchers can determine the three-dimensional structure of proteins and other macromolecules in solution with an accuracy comparable to that of X-ray diffraction. Multidimensional NMR is also being used to study the interactions of biological molecules with other substances, such as water and metal ions, as well as the effects that certain drugs have on metabolic processes. Magnetic resonance imaging, which enables researchers to visualize soft tissues in the body, is also based on the pulse techniques developed by Ernst. According to data collected by ISI, Ernst was one of the 300 most cited scientists from 1981 through 1990. Physiology Or Medicine The Nobel Prize in physiology or medicine was awarded to German scientists Erwin Neher and Bert Sakmann for the development of the patch-clamp technique of recording tiny electrical currents that pass through single-ion channels of cells. Using this technique, Neher and Sakmann proved conclusively that ion channels, which control the movement of ions through cell membranes, do exist, and established how these channels function. Neher directs the membrane biophysics department at the Max Planck Institute for Biophysical Chemistry in G”ttingen, and Sakmann is director of the department of cell physiology at the Max Planck Institute for Medical Research, Heidelberg. Neher and Sakmann first introduced the patch-clamp method to the scientific community in a brief letter to Nature in 1976 ("Single-channel currents recorded from membrane of denervated frog muscle fibers," 260:799-802). In this paper, the authors described a system whereby a micropipette was closely applied to the cell membrane, creating an electrically isolated patch of membrane within the pipette. By restricting the current to be measured to such a small area, they reduced the background noise that previously obscured fine readings. This improvement allowed, for the first time, detection of the tiny signals--measured in trillionths of amps--that are generated when single-ion channels open and close. This paper, according to the SCI, had been cited more than 400 times through 1990. It reported a fundamental breakthrough; yet if development of the procedure had stopped there, instead of becoming the common technique that it is today, "it would have remained a method for people doing heroic experiments," says Frederick J. Sigworth, a Yale University School of Medicine professor who has worked closely with Neher and Sakmann over the years. Sigworth explains that patch-clamp technology really went through two generations, the second marked by the 1981 paper "Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches" (O.P. Hamill, A. Marty, E. Neher, B. Sakmann, F.J. Sigworth, Pflugers Archiv-European Journal of Physiology, 391:85-100). This paper was built upon Neher's 1980 discovery that a very tight seal--dubbed a "gigaseal" by the research group--between micropipette and cell membrane can be achieved if the two surfaces are very clean. This apparently simple discovery led to the radical simplification of the apparently simple discovery led to the radical simplification of the patch-clamp methodology, broadening its number of research applications many times over. In the method first outlined by Neher and Sakmann, says Sigworth, a single current recording was a difficult and complicated process. The cell being measured had to be steadied with a glass hook to control current leakage between the membrane and the micropipette, it was poked with two microelectrodes that measured the current, and the micropipette isolating the membrane patch had to be applied. To control all this, the researcher needed four micro-manipulators hooked up to a single cell. The tight gigaseal (its resistance is measured in giga-ohms, hence the name) obviated most of these props. The seal literally sticks the pipette to the cell membrane, eliminating the need for the glass hook, and the high resistance of the seal--1,000 times higher than the seal achieved in the old method--reduces background noise by an order of magnitude, eliminating the need for the microelectrodes. The importance of this breakthrough can be measured in part by the citation record of the Pflugers Archiv paper describing it: In the 10 years since publication, it has been cited more than 2,600 times. The annual citation rate has climbed each year, topping 700 in 1990, reflecting the fundamental importance of the paper and the rapidly growing scope of patch-clamp technology. Like Ernst, both Sakmann and Neher are among the 300 most cited scientists during the 1980s, according to the SCI. The new, improved patch-clamp system allowed many new applications of the technique. If a micropipette is gently pulled away from the cell to which it is adhered, the piece of membrane within the pipette goes with it, creating a "cell-free" patch. (The cell is not traumatized--it just seals itself back over, says Sigworth.) This bit of disembodied membrane can then be used to test the effects of various substances, such as drugs or enzymes. Such patches have been used to trace, among other things, the signals through which adrenaline speeds up the heart. Cell-free patches have also allowed researchers to observe the effects of neurotransmitters on the ion channels. Channels that respond to a certain neurotransmitter open or close, and the current measured by the patch clamp changes accordingly. Patch-clamp technique also makes possible "whole-cell recording," in which a window to the entire cell is opened by piercing the part of the membrane isolated within the micropipette. Recent advances in cystic fibrosis research, linking the CF gene to chloride channels, utilized whole-cell recording, says Sigworth. The patch-clamp system--and Sakmann and Neher's demonstration of the very existence of ion channels--has since moved into many areas of biological investigation. For example, molecular biologists, trying to understand how channel proteins work, are studying mutated genes in an effort to piece together the underlying genetics. Sakmann is currently a leader in this effort. Neher, although still using the whole-cell recording technique, has, as Sigworth says, "moved away from channels to harder questions." By measuring the electrical capacitance of an entire cell membrane, Sigworth explains, Neher is studying the fundamental mechanics of secretion. When a cell secretes a substance, the membrane of the vesicle in which the chemical is stored fuses to the cell membrane. This enlarges the surface area of the cell, and the resulting change in the cell's electrical properties can be measured. Physics The Nobel Prize in physics was awarded to French physicist Pierre-Gilles de Gennes, whom the awarding committee of the Royal Swedish Academy of Sciences called "the Isaac Newton of our time." De Gennes, a professor at the Coll`ge de France, Paris, "has succeeded in perceiving common features in order phenomena in very widely differing physical systems, and has been able to formulate rules for how such systems move from order to disorder," according to the academy. De Gennes's work brought together theory governing phase transitions in magnets, polymers, superconductors, and liquid crystals. An example of an extremely simple phase transition is that of water changing to ice. De Gennes's work, however, elucidated much more complex systems, such as materials' becoming magnetic or superconducting under certain conditions, or polymers' changing from a liquid to a plastic. Frank Fradin, associate director for physical research at Argonne National Laboratory in Illinois, says that prior to de Gennes's work, "scientists tended to look at each material that underwent phase transitions as a totally different physical problem. They didn't see the underlying commonalities." According to the Nobel committee, while physicists often prefer to work with systems "as simple and pure as possible," de Gennes has shown that "even `untidy' physical systems can successfully be described in general terms." De Gennes has not limited his work to broad brush-strokes, however. He has also "established a microscopic understanding of the behavior of these materials when they undergo phase transformations," according to Fradin. For example, in his work with polymers in the 1970s, de Gennes developed a mathematical model to describe how individual polymer chains move past each other in the spaghetti-like tangle of chains found in concentrated polymer solutions. In this model, called "reptation," the individual chains move lengthwise in a serpentine motion. This explains how some polymer combinations--two tangled piles of spaghetti--can form polymer blends. De Gennes, director of the Ecole de Physique et Chimie in Paris since 1976, began his work on magnetic phase transitions in the 1950s. In the 1960s, he moved on to superconductors and liquid crystals and formed the liquid crystal group at the University of Paris, Orsay, which quickly became one of the leading research teams in liquid crystal theory and related investigations. In 1974, he published The Physics of Liquid Crystals (Oxford University Press), a book that has become a standard work. He formed a polymer group at Orsay--also a leader in its field--when he began studying these materials in the 1970s, and in 1979 published another now-standard work, Scaling Concepts in Polymer Physics (Cornell University Press). While the major contributions of this year's other three science Nobel laureates can be traced to one or two seminal papers, de Gennes's work is more difficult to pin down. "It's really more the body of work than a single paper" that de Gennes is being recognized for, says Fradin. De Gennes's books are classics in the discipline, and both have been heavily cited since they were published. Scaling Concepts was cited nearly 300 times in 1990 alone, and The Physics of Liquid Crystals was cited nearly 150 times in 1990.

1991 Nobel Prize Winners Sparked Fundamental Advances

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