04/13/93
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STANFORD -- For decades scientists have tried to understand how living things work by studying their shapes. They used increasingly sophisticated tools to peek into ever-smaller units of life: tissues, cells, intracellular organelles, macromolecules, such as DNA, or proteins - and have learned something new about biological functions with every biological layer they peeled off.
Now, structural researchers are extending their vision to the atomic makeup of enzymes, aided by accelerator technology that originally was developed for basic science studies in particle physics.
With the help of synchrotron radiation tapped from Stanford's SPEAR storage ring, Keith Hodgson and his co-workers have helped pioneer the development of a technique, X-ray absorption spectroscopy (XAS), to probe the atomic environment around metal components of proteins. It figures out molecular structures by decoding weak rebounding waves - just as a dolphin identifies surrounding obstacles by sending out sound waves.
Synchrotron radiation is extremely intense light that charged particles emit when bent on a circular path at near-relativistic speed.
Hodgson, professor of chemistry, presented new experimental data and theoretical advances of his technique at the annual meeting of the American Physical Society in Seattle recently.
Hodgson also is assistant director of the Stanford Synchrotron Radiation Laboratory (SSRL) that develops synchrotron radiation-based techniques and applies them to many problems in biology, physics, chemistry and medicine. More than 500 researchers from all over the world use SSRL's experimental stations and services each year - and many of them study the structure of biological molecules, mostly enzymes.
Enzymes are the protein workhorses that catalyze the complex chemistry of virtually every process of life, from development to decay. A key to how enzymes work lies in the three-dimensional organization of their atoms and in the distribution of tiny bonds and charges around the enzyme's heart, the active site.
XAS measures the absorption of individual metal atoms to identify their nearest neighbors within a protein. Since such metal atoms often are directly involved in the enzyme reaction, figuring out their spatial arrangement promises clues to how the enzyme does its job.
"With synchrotron radiation as the enabling technology, we can study a specific element in a complex molecule," Hodgson said. "We want to find out the local environment of a single atom. These aren't naked atoms sitting in big proteins; they have things around them - for example, sulfur or iron."
As if peeping through a tiny keyhole, XAS "illuminates" only a very small area of a protein (about 5 angstroms), but does so in tremendous detail. In contrast, a related, widely used technique called X- ray crystallography visualizes the three-dimensional structure of entire proteins, but the resolution is typically only about a tenth of that achievable with XAS.
"Using XAS, we focus just on the information we want, but get that very accurately," Hodgson said.
To obtain such information, the researchers "tune" the initially white synchrotron light to pick out a narrow band of wavelengths. Then they shine it through an enzyme sample, aiming at individual elements by choosing the wavelength at which that element will be excited.
When a sufficiently strong synchrotron beam hits a metal atom, the energy kicks some of the atom's innermost electrons out of its boundaries, propelling them through the neighborhood. There, the propagating particles collide with adjacent atoms, bounce off and create complex patterns of weak electromagnetic waves that the researchers record.
"[These patterns] contain direct proof of the structure within the local environment of about 5 angstroms around the selected atom," Hodgson said.
Analyzing the rebounding waves, the researchers learn what type of atom stood in the way of the traveling particle, where it is and how many of its kind are there. Sometimes, even the bond angle between the atoms is encoded in the recorded waves.
From this electromagnetic hum, the chemists ultimately deduce the arrangement the metal atoms form inside the protein.
In one project, they established the architecture of a "complex" that molybdenum, sulfur and iron atoms build inside a bacterial enzyme called nitrogenase. A natural fertilizer, that enzyme converts nitrogen from its naturally occurring form in the air into a form plants need to grow.
Now that the researchers know the structure, they can study the chemistry happening at this complex, again with XAS. Using "inhibitors" (molecules that stop the reaction midstream) or telltale chemicals in the enzyme reaction, they try to figure out just how nitrogenase transfers electrons to nitrogen.
"Our first challenge was determining the structure, then we tested how it changes with simple reaction chemistry. Finding out how the reaction occurs is where we are heading now," Hodgson said.
Nitrogenase serves as natural fertilizer through a trick of nature: the bacteria harboring the enzyme live in symbiosis with many crop plants. The bacteria form thick lumps in the plant's roots, where they enjoy comfortable protection and, in return, supply their host with an essential nutrient more efficiently than any artificial fertilizer does.
While manipulating nitrogenase may have enormous benefits for agriculture, Hodgson said, "our group is on the basic science side of this. We do not work on new fertilizers, yet a deeper understanding of the processes of the natural systems can guide the design of better catalysts."
Hodgson, who collaborates and publishes extensively with his wife, Britt Hedman, an independent scientist at SSRL, also studies another bacterial enzyme called methane monooxygenase. It normally converts methane into methanol but is promiscuous enough to react also with other compounds, such as complex hydrocarbons found in crude oil and even chlorinated hydrocarbons, notorious environmental pollutants.
"This enzyme is very interesting from the point of view of bioremediation. Getting these bacteria to eat up oil-slicks would be a great way of degrading hydrocarbons," Hodgson said.
Hodgson's group tries to figure out how this enzyme activates oxygen and shifts electrons around during the reaction. They have already determined a center consisting of two iron atoms in the protein and are currently trying to dissect their role in the sequential reaction steps.
One reason why synchrotron radiation makes XAS such a powerful technique is its enormous intensity. It allows the researchers to investigate enzymes in dilute aqueous solutions resembling their natural environment.
Though widely used by structural researchers, the light source was not initially designed to serve them.
SPEAR was built as a colliding synchrotron, in which electrons and their anti-matter counterparts, positrons, race in opposite directions until they smash together. The results of these high-energy physics experiments later earned two Nobel prizes.
In the early '70s, a small pilot project began to apply synchrotron radiation to the study of matter. The SPEAR ring has long since ceased to be useful for high-energy physics and is now dedicated to producing synchrotron radiation for scientists of various disciplines, including pharmaceutical chemists, crystallographers and material scientists.
SSRL has become so popular with scientists that its services are in constant overdemand; "beam time" is the limiting factor. That will change soon. Armed with $8 million from the U.S. Department of Energy, a new beam line to be dedicated to structural studies will be added to SPEAR within the next three years. SSRL receives additional funding for its biological research from the National Institutes of Health.
Beam lines look like long metal tubes tangentially growing out of the storage ring. The synchrotron light travels inside each tube to one to three experimental stations that are protected from the dangerously intense light through an elaborate interlock system.
Although researchers use the workstations on the nine currently existing beam lines round the clock, they still do not fully exploit SPEAR's capacity.
"SPEAR could easily accommodate eight to 10 more beam lines," Hodgson said, "and in addition to the one we are building now, we are planning two more to come. One would help investigate environmentally toxic chemicals to support the environmental studies programs nationwide, and the other one would be dedicated to detecting trace impurities and studying growth processes in semiconductor materials."
This story was written by Gabrielle Strobel, a science writer intern at the Stanford News Service.
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