Toward the Form of Function

Biology's Final Frontier

It's been called the Star Trek of basic research. The Human Genome Project, launched in 1990 by the National Institutes of Health and the U.S. Department of Energy, is a quest for unprecedented knowledge of the fundamental elements of human life. This $3 billion, 15-year mission will take scientists into the far reaches of genetics, where the basic secrets of chromosomal cause-and-effect lie buried in a biochemical web. The ultimate goal of this massive, international research campaign is to determine the intricate layout of the chemical building blocks making up the foundations of every human gene. Given that humans possess at least 100,000 genes, a mass of DNA molecules configured with three billion distinct "blocks," the task is easily the most daunting challenge genetic scientists have ever faced.

When completed around 2005, these scientists hope to have a complete blueprint of the entire human genetic code, a vast assemblage of information detailing the precise order of the chemical units that make each human gene unique. Such a feat, say scientists, will be analogous to determining the sequence of letters making up every book in a large library.

Aside from being one of the first technological triumphs likely to be hailed in the next century, what will such an accomplishment mean? Once the exact order, or sequence, of all three billion genetic units are known, what then? Geneticists readily admit that knowing how a gene is chemically configured, or "sequenced," is only the faintest first step toward knowing how it works, the ultimate goal of all genetics research.

Scientists generally agree that the end of the Human Genome Project will mark the beginning of the really heavy lifting in the protracted battle to understand how DNA and other molecules do the incredible things they do within the confines of living things. With the complete genetic "map" in hand, the job becomes one of "reading" the vast, newly ordered blueprint and making sense of it all. But hopefully by then, the job will be somewhat easier, thanks to impressive gains now being made in a young field known as structural biology.

An outgrowth of the familiar-sounding field of molecular biology, structural biology is now recognized as the key research specialty setting the pace for advancement in a host of high technologies, from bioengineering to gene therapy. Biological research centers the world over are rapidly beefing up their profiles in the field amid near-daily news accounts of remarkable developments stemming from discoveries made in structural biology labs.

Florida State is no exception. In 1990, the university's Institute of Molecular Biophysics (IMB), formally organized a structural biology program, the institute's newest initiative in the past decade. Jump-started with a $4 million gift from the Miami-based Lucille P. Markey Charitable Trust, the program-with seven full-time faculty researchers-now is the largest program of its kind in the Southeast.

Also helping the new program jump directly into the fore of formidable competition coming from more well-established research centers around the country is its close tie with the National High Magnetic Field Laboratory, headquartered at nearby Innovation Park in Tallahassee. This $100 million enterprise, in full operation since 1993, is equipped with some of the latest and most powerful diagnostic tools in the world, machines enormously important for structural biology research.

"The driving force behind structural biology is the fact that it is very difficult to understand how a complicated machine works if you can't see a picture of it working," Makowski says. "We can now produce that picture, down to the level of every single atom, and use it to focus on questions that can make a difference in people's lives."

Though it didn't get its name until the late 1950s, structural biology is a direct descendant of the startling 1953 discovery of the three-dimensional (3-D) shape of the DNA molecule. Two Cambridge University researchers, James Watson and Francis Crick, awed the scientific world that year when they unveiled the exquisite symmetry of the moledule's gracefully coiled, double-helix structure.

For the first time, biologists had clear, unshakable evidence that the physical, 3-D structure of life's most important molecule plays a profound role in its crucial and extremely varied functions. Scientists soon began to see direct links between the shapes and sizes of all DNA-based molecules and the behavior of the microscopic "organs" -such as cells and cell parts-that these molecules make up in plants and animals.

With tools available to them today that Watson and Crick might never have imagined, structural biologists are adding layer upon layer of knowledge to a growing body of insight into the relationship between the 3-D form of biological molecules and their function. The young human genome effort, with its thrilling, fallout discoveries of such things as faulty genes responsible for a number of human ills, may have grabbed most of the headlines, but the fact is that structural biology is playing equally important roles in many other avenues of research.

At FSU, most of the focus is on determining the 3-D characteristics of the large molecules which serve as the building blocks for such things as cell membranes, muscle fibers and viruses. Just as a house is dependent on its foundation, walls, and roof to serve its function, so do each of these structures depend on an intricate framework of interlocking molecules that governs its development.

Most of the molecules under study at FSU, and indeed in most structural biology research, belong to a special group called macromolecules, so-named because of their enormous sizes. Since life itself is based on a macromolecule-DNA-it's not surprising that almost all of the complex organic chemicals necessary for life-primarily proteins-belong to this family of molecular giants, some of which have upwards of 100,000 atoms embedded in their molecular skeletons.

Since proteins are the engines that drive every biological process imaginable, even from its earliest beginnings structural biology has been focused on a quest to link proteins' 3-D frameworks to their myriad functions. Indeed, whatever gains come from the Human Genome Project are likely to be measured by what scientists learn about the way gene sequencing dictates proteins' structure. How genes are sequenced determines everything about how proteins are built, which in turn determines how they drive everything from cell division to thinking. Last year, FSU hired a scientific pioneer who has spent most of his career studying protein structure, and particularly how it figures into the creation of what is arguably mankind's sturdiest foe-viruses.

Dr. Donald L.D. Caspar (Ph.D. Yale), left a post at Brandeis University to join IMB's new Program in Structural Biology. A member of the National Academy of Science, Caspar is an internationally recognized figure in structural biology, and in fact is credited with giving the field its name (see related article). His work on virus structure is generally regarded as having established the fundamental principals underlying these fascinating organisms' molecular architecture, Makowski said. Makowski feels that the presence of Caspar not only helps put IMB on the scientific map but also goes a long way toward attracting top-flight researchers. Just this fall, the IMB team drew to FSU another prominent specialist in determining protein structure, Dr. Kenneth Taylor (Ph.D. UC Berkeley), formerly of Duke University. Taylor studies the complex mechanisms in the proteins that make human muscle function possible, using electron microscopes to obtain 3-D images of the protein's structure. Taylor is a highly skilled specialist in electron microscopy. Caspar, in turn, is an innovator of uses for the most powerful X-ray machines on earth-giant electron accelerators called synchrotrons -in studying the protein labyrinths of viral structure. Both scientists typify the special calling that marks all structural biologists-these are scientists who are entirely dependent on the availability and power of highly specialized instruments that make their work possible.

The challenge, of course, is to be able to "see" structures that are nothing but strings of atoms. Even the strongest light microscope is of little use in determining the shape and movement of molecules, so biologists must rely on three principal techniques: X-ray crystallography, in which crystallized bits of organic matter are bombarded with X-rays (see box above); nuclear magnetic resonance (NMR) spectroscopy and electron microscopy. When Watson and Crick discovered the double-helical structure of DNA, some of these techniques were around, but none had the sophistication of the tools available today.

So it is that structural biologists have become more or less divided into the camps of the techniques at hand. Such instruments literally allow scientists to take visual "tours" of macromolecular architecture, "seeing" individual atoms and their connections with other atoms, and, on a broader scale, seeing close up the innermost workings of living tissue such as cell nuclei and muscle fiber.

FSU's Taylor, for example, is using his skills in electron microscopy (EM) to develope new techniques for producing 3-D images from a single specimen of living tissue. Already to his credit is a novel, now widely used technique of studying such specimens that eliminates one of EM's major drawbacks in such work. Traditionally, EM users faced a dilemma: To study living tissue, they literally had to embalm it to observe it in the ultra-high vacuum of the electron microcope. Water, the essential component of all living systems, was usually replaced by inorganic "stains" to provide a non-volatile supporting medium. Taylor developed a technique in which the sample is encased in ice, creating a gentler process that maintains the molecules in as near their living condition as possible. The planned arrival next spring of a new high-powered electron microscope to the institute's on-campus home is another IMB coup. Even with Taylor's innovative ice technique, EM can be a dicey tool for biologists, because the electrons these machines fire at targets can damage specimens. Microscopes generating electron-beam energies of only 100 kilovolts (KeVs), for example, can tear such specimens apart. At 200 KeVs, the damage is less, and it's less still at 300 KeV, the power of the machine scheduled to be installed within IMB. Its arrival, along with a field emission gun (essentially an electron laser) aimed at studying atomic contrast at the finest level of detail, will make FSU-based EM second to none in the business, Makowski says.

For sophisticated X-ray work, FSU researchers such as Caspar aren't likely to find an instrument they can use in Tallahassee any time soon. Because of their enormous cost, ultra-high-powered sources of X-rays are confined primarily to national facilities such as Brookhaven National Laboratory in New York. Such machines are highly useful in examining the molecular structure of biological specimens in crystallized form.

If scientists can get a specimen to crystallize, they can bombard it with high-powered X-rays and hope to get a detailed pattern of X-radiation that gets reflected off atoms making up the specimen's molecules. Such patterns can tell researchers precisely where individual atoms lie along a m olecule's framework, indispensable information for determining its shape. But it may be NMR spectroscopy that puts IMB's structural biology research a notch ahead of the competition. The close relationship with the magnet lab gives IMB access to some of the best, most cutting-edge NMR facilities available anywhere, Makowski says.

"The NMR facility here is among the best in the world," echoes Dr. Tim Logan (Ph.D. Chicago), who uses it to study how and why proteins fold into various 3-D structures, the most vexing problem in the field. "There's a great core of people there, the support staff is excellent, and they have some of the best machinery in the world."

That the magnet lab now boasts such a facility is a testimony to the work of such FSU researchers as Dr. Tim Cross, a chemist and ardent champion of using NMR techniques in structural biology. When word first spread of FSU's decision to vie for the federal grant to establish the magnet lab in 1990, Cross collaborated with Dr. Jack Crow, now NHMFL director, on the proposal to the National Science Foundation. That proposal included a provision for a 27,000-sq.-ft. facility dedicated exclusively to exploiting NMR's potential for use in physics, chemistry and particularly in structural biology. Cross, who served as the structural biology program's first director, now is director of new intiatives within the NHMFL. But for all their power and sophistication, the tools in the hands of today's structural biologists seem almost crude when one considers the questions posed by even the simplest living organism. How is it that only a few lifeless elements-mainly oxygen, nitrogen, carbon, hydrogen and phosphorous-can come together in electrifying ways to create life? How can it be that seemingly lifeless molecules move? Why do identical genes work in some places and not in others? Such questions are hardly new, and some seem as intractable today as they ever did. However, recent advances suggest promising new approaches to reliable answers.

If nothing else, the rise of applications in biotechnology, a field unheard of just 20 years ago, has demonstrated the value of basic research in structural biology. Sick people are being cured today with manmade drugs built literally an atom at a time, thanks to fundamental knowledge of molecular structure and behavior. From gene research has sprung gene therapy, which, although still in its infancy, is a branch of medicine that many think will revolutionize the diagnosis and treatment of hundreds of genetically based diseases.

IMB researcher Dr. Michael Chapman (Ph.D. UCLA), is one of many structural biologists today intrigued by the idea of using viruses, for example, as therapeutic agents for fighting cancer. He studies the atomic structures of harmless viruses that might be used as vectors, carrying good copies of damaged genes and depositing them wherever the body might need them. The idea is that if a virus can be engineered to carry a good copy of BRCA1-the gene that researchers have found predisposes women to breast cancer-and drop it off at the right chromosome without being attacked by the human immune system first, physicians would have a good chance at stopping breast cancer before it starts. If they could use this same technique on the gene that causes muscular dystrophy, adult carriers of this debilitating killer could arrest it before passing it on to their children.

Chapman also studies the atomic structure of a relative of the human enzyme creatine kinase, which is responsible for replenishing energy resources inside such things as heart muscle. Physicians measure for presence of the enzyme as a way of determining whether a patient has had a heart attack. Better knowledge of the enzyme's atomic structure will help researchers design a more reliable clinical diagnosis, Chapman says.

"What all of us are trying to do here," he adds, "is to use an understanding of molecular structure to improve health." Poised to tackle a broad range of problems with both excellent facilities and scholars, the IMB has generatedan environment that appeals not only to its faculty researchers, but to would-be researchers as well, says Makowski. The place is abuzz with the enthusiasm of new projects designed to engage both teacher and student.

"Research is really just the practice of learning, of thinking in a highly disciplined way," added Makowski. "Since our main goal is to teach our students to think and to learn, I believe that all students should participate in research."

The mix of missions-teaching and research-is well-balanced within the IMB, Caspar feels, another reason he's convinced the program is a good investment for both FSU and the field of structural biology.

"Resources have gotten much more strained in science during the past decade," Caspar notes. "As a result, a lot of the projects in structural studies at other places have rather narrow focuses.

"Here at Florida State, we've got an exceptional opportunity to build up a new, broad-based program. There are going to be opportunities here to do novel work in a number of exciting directions."