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| 78.4 - Summer 2005 | ||||||||||||
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Probing the Building Blocks of Life
Innovative tools add a new dimension to structural biology |
Printable Version |
By Nina Bai
More than two millennia ago, armed with scientific curiosity and keen observation, Aristotle refuted the then widely-held belief that elephants had no joints; three hundred years ago, with the aid of a simple light microscope, Anton van Leeuwenhoek first described muscle fibers and capillary blood flow; and fifty years ago, applying the still fledgling technology of x-ray crystallography, Rosalind Franklin found clues to the helical structure of the DNA molecule. On some level, each of these individuals can be considered the structural biologists of his or her time, expanding our knowledge of the building blocks of life. Today, with high-resolution electron microscopes, powerful synchrotron light sources, and potent computers, Yale’s structural biologists are further probing the structures of the most intricate proteins and nucleic acids.
X-ray crystallography uses the scattering of x-rays by electron clouds of atoms to give give electron density information. (Credit: NASA)
“Structural biology” encompasses the study of biological macromolecular structure, including that of protein, DNA, and RNA. Unveiling the structure of these macromolecules will allow scientists to determine their function and properties. As Sterling Professor of Molecular Biophysics and Biochemistry Thomas Steitz explains, today’s structural biologists are breaking down the barrier between two disciplines as they look for the “chemical basis of biological function.” In the quest to understand the mechanisms by which an enzyme functions, a receptor binds, or a protein folds, “the explanations are [often] straight chemistry.”
Gotta Have the Right ToolsThe rapid advancement of the field of structural biology in recent years has followed the trajectory of new technology. The three main methods of molecular structure determination—NMR spectroscopy, electron microscopy, and x-ray crystallography—operate on different principles but complement each other. Developed in the 1940’s, nuclear magnetic resonance (NMR) spectroscopy, relies on the interaction of magnetic fields with the spin of atomic nuclei. Data from NMR spectroscopy reveals the identity and relative orientation of atoms in a molecule.
X-ray crystallography uses the scattering of x-rays by the electron clouds of atoms to give electron density information. Atomic structure and covalent chemistry can be determined using this x-ray diffraction data. Sterling Professor of Chemistry Peter Moore explains, “Over my career, crystallography has developed from its primordial beginnings to a rather mature tool for determining the structure of large molecules.” The power of x-ray crystallography has increased tremendously in recent decades thanks to the development of synchrotron light sources. Synchrotron radiation, which is emitted when charged particles are forced to move in a circular orbit close to the speed of light, was originally regarded as an unwanted byproduct of high energy particle accelerators. Since the 1970’s, synchrotron radiation has been adapted for use in x-ray crystallography because of its extraordinary brightness and wide energy spectrum. While providing ultimate resolution, x-rays are not suitable for recording direct images; therefore, x-ray crystallographers require highly ordered, crystalline samples whose diffraction patterns provide a handle for structure determination. With the aid of computer programs and biochemical predictions, electron density information can be reconstructed into 3D molecular models.
A relative newcomer to the structural biology stage is electron microscopy (EM). While its usefulness was recognized around the same time that x-ray crystallography began solving the first protein structures, many technical hurdles prevented its widespread use. Only in recent years has the resolution of electron microscopes approached the atomic level, yet it remains limited to the analysis of 2D crystalline arrays. However, the visualization of protein structure at lower resolutions also has its benefits. “Very few molecules are individual players in living cells; they all work together,” explains Vinzenz Unger, associate professor of molecular biophysics and biochemistry, who uses EM to study membrane proteins. Sometimes, a glimpse of the overall structure is a helpful starting point for understanding how the many macromolecular machines inside our cells carry out their function. EM can reveal the basic shape of these molecular assemblies and is particularly useful for working with flexible macromolecules that are difficult to prepare for x-ray crystallography.
Advancements in the preparation, analysis, and computation of molecular structure have come a long way. “When I was a student in the ‘60s,” Steitz recalls, “we used whatever was abundant in the cow pancreas. Now we have the tools to make the materials and are able to clone large quantities.” Even with these improvements, structure determination is still a lengthy process—a test of patience and perseverance often taking many years. Isolating and crystallizing molecules can be tedious and slow. With the ultimate goal of understanding how these molecules function, one must carefully isolate the molecules at the appropriate stages in biological pathways. “The problem is capturing the molecule in the process,” explains Steitz, whose lab recently unraveled the mechanism of the CCA-adding enzyme (tRNA nucleotidyltransferases) by taking x-ray crystallography “snapshots” of the enzyme at work (see also “The Smart CCA-Adding Enzyme” Yale Scientific, Vol. 78, Winter).
The Structure-Function RelationshipThe path from structure to function is traversed with the help of genetic and biochemical information. “Structural biology is useless by itself. Structures are most useful when they can be related to other information,” explains Moore. The physical structure of a folded protein cannot by itself reveal how the protein functions, since the 20 amino acids that make up proteins can combine in more ways than there are atoms in the known universe, making the task of predicting a protein’s function dauntingly difficult.
In the past, chemistry was used to hypothesize structure. As structural determination has reached new heights, however, structure has become the basis for studying the mechanisms of chemical reactions. Structures that look alike, act alike. Energy calculations coupled with experimental data are used to make chemically and thermodynamically plausible inferences about how a molecule functions. These inferences are then tested, often by making genetic mutations or deletions of parts of the molecule and analyzing the subsequent changes in behavior.
In the background are electron micrographs of Salmonella needle complexes. The foreground image is a computer-generated 3D structure of this complex. (Credit: Vincenz Unger)
Structural biology research abounds with applications. The study of macromolecules will elucidate basic life processes and inspire new and better drug design to treat complex diseases.
Over 24,000 protein structures are currently known and stored in the Protein Data Bank; yet, only about sixty of these structures are membrane proteins. Considering that membrane proteins make up nearly thirty percent of all genes and seventy-five percent of the genes targeted for drug develpment, this huge discrepancy reflects the difficulty of crystallizing such proteins. Unger’s research falls in precisely this catergory: in one example, the Unger lab has been studying a transmembrane protein involved in cellular copper uptake and the transport of cisplatin, an important anticancer agent. Recently, the lab has also studied the structure of secretory needle complexes in the bacterium, Salmonella typhimurium. Understanding the structure-function relationship is essential for investigating the virulence of such bacteria.
On the other hand, Steitz has had much success in establishing structures of the macromolecules involved in all steps of gene expression. “We want to study all the enzymes involved in the Central Dogma” states Steitz, referring to the model of genetic information flow from DNA to RNA to protein. Steitz’s work on ribosome structure may aid the design of drugs to overcome antibiotic resistance. Approximately 1.4 million people will contract an antibiotic-resistant infection in a US hospital this year. Many antibiotics specifically target bacterial ribosomes. By mutating in such a way that they no longer bind to a drug’s active site, bacteria and viruses can develop resistance to that drug. Thus, structural information on the ribosome will be key in designing new antibiotics.
As most researchers would agree, the next challenge is to look at very large biological molecules. These include complex macromolecular assemblies such as ribosomes and replisomes. Moore predicts that as the analytical tools become more user-friendly and widely available, the field of structural biology will be one in which “anybody can play.”
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