The following is excerpted from a March 1996 Penn Health Magazine article by Franklin Hoke incorporating material by Esaúl Sánchez.
The research team is composed of scientists from the Department of Biochemistry and Biophysics at Penn's Medical Center, led by Mitchell Lewis, M.A., D. Phil., and from the Department of Chemistry in the School of Arts and Sciences, led by Ponzy Lu, Ph.D. Two other researchers are from Oregon Health Sciences University.
The object of this long search is the protein responsible for regulating a cluster of genes that control the metabolism of lactose in Escherichia cold bacteria. Now that they have a clear picture of the protein's threedimensional structure in hand, scientists can understand precisely how the lac repressor functions at the molecular level. One possible result is that researchers may now be able to customize similar molecular switches to turn selected genes on or off on demand. Such a technology would have significant implications for gene therapy and other molecular medicine.
"You can imagine taking the guts of this repressor and reengineering it to recognize molecules other than this particular sugar or DNA sequence," says Dr. Lewis, associate professor of biochemistry and biophysics and lead author on the study. "You could redesign the repressor to respond to a specific drug. You would then be able to turn a gene or set of genes on or off by administering that drug. And I don't think that's far down the road. In fact, that's what we're hoping to do next."
The research effort led by Drs. Lewis and Lu places the capstone on three decades of illustrious molecular biology. In the late 1950s, two French scientists, François Jacob and Jacques Monod, described the regulatory role of the lac repressor. In 1965, they won the Nobel Prize for their work. Another Nobel Prizewinner, Walter Gilbert, discovered in 1966 that the repressor was a type of protein, and its 360 amino acids have subsequently been identified and fully sequenced.
In the years since then, however, scientists had tried in vain to determine the lac repressor's shape. As Dr. Lu has explained, before scientists can design and synthesize drugs to fight diseases, they need detailed information about the surface shape and interior geometry of proteins: "Knowing the protein's chemical composition is not enough, because molecules with identical composition can have a different structure, and this usually leads to a different behavior. Surface contours and interior geometry determine which molecules can activate a protein and what the protein can do afterward."
Because proteins are small in size, existing microscopes cannot pick up the nuances of a protein's shape. What scientists must do is "pack" together millions of the same proteins into an ordered solid, called a crystal, and then use Xray diffraction to deduce its structure. In this procedure, the crystals are bombarded with Xrays; the patterns made by the Xrays as they bounce off the atoms in the protein crystal are recorded on film. Next, these patterns are analyzed by what The New York Times, in an article on Drs. Lewis and Lu's achievement, called "complicated mathematical and computational methods"; then the scientists reconstruct the shape of the crystal.
In the case of the lac repressor, this crucial information would allow them to understand the molecular mechanism of gene regulation in a typical system. Yet this same protein presented unusual problems and would not crystallize well. At one point, about three years ago, the team tried a novel approach: they sent their crystals into space aboard a space shuttle. The hope was that without the pull of gravity, the "seed" of the crystal would remain in the middle of its flask, allowing proteins to attach anywhere around it, thus forming larger crystals...To the dismay of the Penn researchers, however, even the crystals "grown" in space proved too small for use.
Finally, in what The New York Times characterized as "an arduous feat that involved luck as well as dogged persistence," Drs. Lewis and Lu were able to make the crystals. One of the steps involved adding a nickel compound to the molecules that helped them analyze their data.
As a result, their laboratories were able to crystallize the lac repressor in three conformations: bound to DNA, inactivating the lac genes; bound to the sugar, allowing transcription of the genes; and unbound to any other molecules. "This is the first detailed picture of how gene regulation occurs at the molecular level," Dr. Lewis says. "That's why it's exciting, both historically and scientifically."
Volume 42 Number 30
April 30, 1996
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