Although X-ray crystallography might sound incomprehensible to some, researchers in the Molecular Biophysics and Biochemistry (MB&B) Department are using the technique to understand how biology operates at the molecular level, and to use this knowledge to discover exciting applications in modern medicine.
X-ray crystallography is one of two methods which allow researchers to determine the structure of macromolecules such as proteins and DNA. Knowing the structure of these key molecules often provides valuable insights into the way they function, paving the way for drugs designed to interact with them.
Professor of MB&B and Chemistry Thomas Steitz uses X-ray crystallography to "solve the structure" of key human enzymes, particularly those which interact with DNA or RNA. The crystal structures that he and his laboratory have solved include: glutaminyl-tRNA synthetase, an enzyme involved in protein synthesis; DNA polymerase I, an enzyme involved in DNA replication and repair; and HIV reverse transcriptase, an enzyme which allows the HIV virus to replicate and infect host cells.
Steitz and other investigators in his lab are currently trying to understand the mechanism by which reverse transcriptase activates the propagation of HIV. "We're working on solving the structure of this protein with a piece of the viral RNA to which it binds," Steitz said.
The process of solving a protein crystal structure begins by obtaining large amounts of the protein to be studied. With modern genetic techniques, researchers can use bacteria or other microscopic organisms as protein factories, literally tiny machines capable of producing large amounts of a specific protein. The protein is then purified from the bacteria and used to grow crystals, a tedious process often requiring years of effort.
If a crystal is obtained, it is analyzed by X-ray diffraction. X-rays are passed through the crystal, and each atom in the protein causes the rays to diffract differently. This diffraction pattern is then recorded by a sensitive detector. About 10 to 100,000 measurements are made in different areas of the crystal, allowing the researcher to gain a complete picture of its structure. After a series of complex calculations, the result is a beautiful photograph of the protein in three dimensions, which can be displayed and manipulated on a computer.
Using X-ray crystallography, researchers in Steitz' lab have discovered the molecular site where HIV is manufactured. The virus contains RNA, which is a single-stranded molecule that holds genetic information essential to the virus' replication and its takeover of a cell's machinery. HIV reverse transcriptase recognizes a region of RNA and binds to it. This activates a process in which the virus' DNA is manufactured using its RNA as a template. Using x-ray crystallography, Steitz' lab has solved the structure of the resulting DNA-protein complex.
"Once you solve the structure of this complex, you can then design ways to interfere with this process," Steitz said. "Understanding the structure helps reduce the number of inhibitors one might use to break down the process that initiates HIV's replication." In this way, crystallographic studies have helped researchers actually design drugs that can interact with macromolecules in specific ways.
Steitz added, however, that the main purpose of X-ray crystallography studies is to understand fundamental biological processes, and is not necessarily a stepping stone toward rational drug design. Structural biology often provides insights into molecular evolution, he said. "There are striking relationships between different protein structures that would not be otherwise noticed through the amino acid sequence," which is the sequence of building blocks that makes up a protein.
One can appreciate the sophistication of today's X-ray crystallography methods by looking at how far researchers have progressed since the first protein crystal structure was solved in the 1960s. After 30 years of working in structural biology, Steitz said he has seen tremendous changes in the way students collect diffraction data. "As a graduate student," Steitz said, "it took a year for seven graduate students to collect data on a protein called carboxypeptidase. A couple of days ago, one single graduate student took data for an enzyme of similar size in 12 hours."
Steitz said that when the pioneers of his field solved structures of proteins, they built models out of copper wires. Today, a computer can display three-dimensional models of these structures with little effort. In addition, better techniques to purify proteins have allowed one to look at virtually any protein, he said.
In addition to Steitz, other Yale researchers are attempting to solve different problems in structural biology. At the medical school, one group is trying to elucidate the structures of several proteins which are involved in the human immune system.
Professor of MB&B Paul Sigler is working with a type of protein called a transcription factor, a general class of proteins that regulates the decoding of DNA into proteins. Sigler is working with transcription factors responsible for the regulation of "killer proteins," which come to the immune system's rescue when a virus invades the body.
According to Gourisankar Ghosh of the Sigler lab, X-ray crystallography is critical for understanding how these transcription factors work. When a harmful virus or bacteria invades the human body, a signal activates the protein that helps initiate transcription by binding to DNA. Ghosh said that he and Sigler use X-ray crystallography to understand the interaction between DNA and the protein. "Without x-ray crystallography," Ghosh said, "there would be no way to imagine this protein-DNA interaction." A clear picture of this complex makes drug design possible, he said. "We have to know the structure to design any individual drugs."
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