Building On Firm Foundations: Structural Biology Is Coming Of Age

Coming Of Age Structural biology--the study of the three-dimensional shapes of biological macromolecules--has always been a facet of biological research, from the detection of cells in the earliest days of microscopy in the late 17th century to the first X-ray crystallographic images of proteins in the 1950s and the development of DNA technologies that made proteins more accessible for study through the 1970s and 1980s. BUILDING BLOCKS "Every biological process occurs within, or is associated with, some structure," says Kenneth Miller, a professor of biology at Brown University, Providence, R.I. And in order to truly understand these processes, he says, there is no substitute for knowing the molecular details of that structure. Miller and other researchers agree that proteins--more than any other macromolecule--are the functional units of biological activity. Some of the more important proteins that are studied by structural biologists are: enzymes that catalyze such vital processes as DNA replication, respiration, energy-transfer reactions, and photosynthesis; receptor molecules on cell membranes; and antibodies that ward off infections. The field of structural biology primarily focuses on elucidating the three-dimensional structures of proteins. While such other macromolecules as DNA and RNA are also fundamental to living systems and processes, structural biologists point out that the linear sequence of these nucleotides, rather than their three-dimensional configuration, is the most important aspect of their structure. "I like to think of DNA as the one-dimensional repertoire of information for producing the three-dimensional information for proteins," says Edwin Westbrook, director of the new APS Structural Biology Center at Argonne National Laboratory. "To use a theological analogy, it's like the Word made flesh." Retrieving DNA sequence information is now a standard technique, with large national efforts such as the Human Genome Project routinely using the procedure. Researchers also point out that obtaining the amino acid sequence of a protein is only the first step in determining a protein's structure. The basis of function, they say, lies in the three-dimensional conformation of the molecule; for example, an enzyme molecule can be rendered active or inactive just by changing its shape. Thus, a structural biologist's task is to dissect the details of protein conformation--how it is folded, the relative positions of atoms in the molecule, and the nature of hydrogen bonds between amino acid residues not adjacent to each other. Using such sophisticated techniques as crystallography and nuclear magnetic resonance, scientists are now conducting detailed analyses on the atomic-level structures of various proteins. But over the past 10 years, scientists agree, this field has been experiencing a boom. Its growth is evidenced by the impressive proliferation of protein crystallographic data and research being produced by various segments of the biological community, spurred by technological breakthroughs from a wide array of disciplines. The forums and facilities sprouting up to accommodate this flood of information and interest are further indications of the discipline's rapid emergence. "Structural biology is now at the stage DNA research was at about 50 years ago," says Guy Riddihough, editor of Nature Structural Biology, one of two peer-reviewed journals devoted entirely to the subject that were created in the past year to keep up with the volume of new information. Structure, published by the London-based Current Biology Ltd., was launched in September 1993, and Nature Structural Biology--an offshoot of the journal Nature and published by Macmillan Magazines Ltd. in London--came out this past January. In just the past three years, Brookhaven National Laboratory's Protein Data Bank (PDB) in Upton, N.Y., has doubled the amount of data on protein structures it has received from researchers throughout the United States, PDB officials say. PDB was established in the early 1970s by the National Science Foundation to collect and store such data. It is now funded jointly by the U.S. Department of Energy (DOE), the National Institutes of Health, and NSF. "The number of protein structures [PDB is] receiving is rising exponentially," says Edwin West- brook, director of the new Advanced Photon Source (APS) Structural Biology Center at Argonne National Laboratory (ANL) in Illinois. "They are having trouble keeping up." DOE is spending nearly a half-billion dollars to build APS, a circular--more than a half-mile in cir- cumference-- synchrotron X-ray facility that will produce high-intensity X-rays. DOE has invested a further $15 million to establish the APS Structural Biology Center at the lab, which will use the synchrotron's X-rays for various structural studies. APS resources will also be used by two other structural biology research centers currently being constructed nearby: one by the University of Chicago and the other by a newly created consortium of pharmaceutical firms called the Industrial Macromolecular Crystallography Association. Modern structural biology focuses on the three-dimensional configurations of macromolecules--in particular the proteins and nucleic acids--that make up the cells and perform various important biological functions such as DNA replication, photosynthesis, and the formation of antibodies (see accompanying story). David Davies, an X-ray crystallographer and a researcher at NIH, says that the discipline really took hold in the 1930s and 1940s with the application of X-ray diffraction to obtain the shapes of molecules from crystals. In 1953, James Watson and Francis Crick, using crystallographic data and molecular modeling, proposed the structure of DNA, which revolutionized the field of genetics. According to Wayne Hendrikson, an editor at Structure, this first determination of a macromolecule's configuration ushered in the field of modern structural biology. Also during that time, British crystallographers Max Perutz and John Kendrew began to produce the first structures of proteins such as hemoglobin and myoglobin. But structural studies on proteins proceeded at a modest pace, until, fueled by technical advances in X-ray crystallography, nuclear magnetic resonance (NMR), recombinant DNA technology, and computing, it has come into full flower over the last decade. In addition to the technological achievements that have facilitated continuing breakthroughs in structural biology, the field has been driven by advances in other disciplines, such as physics, chemistry, biochemistry, and biophysics, researchers say. For instance, the guiding principles of methods like X-ray crystallography and NMR, required for determining structures at the atomic level, are rooted in physics and chemistry. X- ray crystallography uses the diffraction patterns of high- intensity X-rays bounced off protein crystals to determine the locations of the atoms within the crystals. NMR spectroscopy--a more recent technique--is used to determine the structures of proteins that are still in solution. "NMR was developed as a method to [determine] protein structures in the late 1970s," says Gerhard Wagner, a professor of biological chemistry and pharmacology at Harvard Medical School in Boston. "Essentially, each hydrogen nucleus [in the molecule] has a characteristic resonance at a particular frequency, depending on the atoms around it." The measurement of these various resonances gives the distances between different nuclei, enabling the three-dimensional structure of the molecule to be calculated, he says. Adding to the value of these techniques in structural biology has been the field of molecular biology, which has seen considerable progress of its own in the past few decades. "Recombinant DNA technology, expression vectors, and the ability to produce almost any protein in large amounts opened the doors to a lot of proteins," says Johann Deisenhofer, a 1988 Nobel laureate in chemistry who is an investigator at the Howard Hughes Medical Institute Research Laboratories at the University of Texas Southwestern Medical Center in Dallas. With the capability of producing large quantities of proteins, scientists have been able to try more approaches to growing crystals. "What drove structural studies earlier was the availability of large amounts of proteins and the ability to crystallize them," says Riddihough. "Crystallization of proteins is a lot like cookery--there are very few rules." Molecular-biological tools, such as some enzymes, have also helped to overcome some of the limitations of NMR in determining protein structure, says Wagner. The maximum size of a protein that can be examined with NMR is 30 kilodaltons; many proteins are considerably larger than this. "We now have strategies to cleave larger proteins into structurally and functionally intact subdomains that can be readily studied by NMR," he says. Coming Of Age Wagner notes that "crystallography and NMR are now mature techniques that students and postdocs learn as a matter of course, rather than approach as goals in methods research. Where previously people with physics backgrounds focusing on methods development were dominant [in crystallography and NMR laboratories], there is now a new class of scientists [who are proficient in these techniques] with a focus on biological problems." "When I began as a graduate student in Oxford, in 1949, it typically took us about one year to determine the structure of a 12-atom molecule," recalls Davies. These molecular-imaging technologies have undergone substantial refinement since then, he says, not just in terms of speed, but also with respect to the quality of information. "With higher-intensity X-rays now being produced in various synchrotrons around the world, we can obtain crystallographic information with much more resolution and rapidity," Riddihough says. "Technical advances have had a quiet but tremendous effect on the progress of the field," says Deisenhofer. "Data collection [measuring the various diffraction lengths] is fully automated now, and the same computer that cost $3 million when I was an undergraduate is now about $1,500." Advances in computer science have been tremendously important in bolstering structural research--both as devices to collect and speedily analyze imaging data, and as tools for modeling molecules, say researchers. Scientists say that the current explosion of research is only the beginning and in the years to come structural biology will gain even more importance. Riddihough predicts: "In a few years people will be taking the products of this research for granted, just as we do [DNA] sequence information now."

Building On Firm Foundations: Structural Biology Is Coming Of Age

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