June 20, 2002 ITHACA, N.Y. -- A newly established national biomedical center at Cornell University is reporting its first major advance: a new way of measuring, or "visualizing," proteins. The new technique will hasten the transformation of the human genome project's blueprints of life into a comprehensive view of the biochemical and physiological circuitry that interconnect to form entire organisms.
The technique, which determines the structure of a protein by measuring the distances between atoms in the molecule at greater separations than previously possible, is an important development, says Jack Freed, professor of chemistry and chemical biology at Cornell, who is director of the National Biomedical Center for Advanced ESR Technology (ACERT), established at Cornell last year by the National Institutes of Health. "This is in the spirit of seeing the whole forest of the protein, whereas before we have been seeing the trees one after another," says Freed.
Freed and his collaborators, Hassane Mchaourab, professor of molecular physiology and biophysics at the Vanderbilt University School of Medicine, and Peter Borbat, associate director of ACERT, report on the new method for protein structure determination in JACS , the Journal of the American Chemical Society (May 22, 2002).
"This technique is potentially very powerful for the investigation of larger protein assemblies and membrane proteins," says Yeon-Kyun Shin, associate professor of biophysics at Iowa State University and a major user of the ACERT facility.
The new method for seeing the structure of the protein uses ESR (electron spin resonance), a technology for studying the bonds, structures, and molecular mechanisms of chemical and biological materials, such as membranes and proteins. Basically, the technique elucidates how molecules move, react and interact with one another. The protein studied for the JACS report, T4 Lysozyme, is one of the proteins of a bacteriophage, or virus, that is parasitic within a bacterium. The protein degrades the bacterial cell wall to enable the virus's exit.
Previously, Freed's group pioneered technology that enables ESR methods to unravel the complex dynamics of biosystems such as proteins and membranes. The research group has adapted this technology, dubbing it DQC (for double quantum coherence), to deliver pulses of microwave radiation in appropriate sequences in order to measure the distances between two spin labels. These are molecular subunits, each containing an unpaired electron, inserted at precise sites in the protein. DQC-ESR "interrogates" the spin labels for their weak interaction, the magnitude of which depends on the distance between them. By measuring such distances, the overall structure of the protein can be revealed.
Until now, protein structure has been determined primarily by two widely used methods: X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. The X-ray method, however, requires crystallization of the protein, and as Freed explains, a protein is not just a single crystal or a frozen object but is in constant flexing and tumbling motion. NMR visualizes the molecule in its normal environment and is based on measuring many small distances between adjacent, or nearly adjacent, atoms, like going from tree to tree. The new technology reported in the JACS paper, which needs only very small amounts of protein, gives researchers a comprehensive view of a the molecule, "like being able to see the topology of the entire forest," says Mchaourab.
He notes that 30 percent of the proteins encoded by a genome and 50 percent of pharmaceutically important receptors are membrane-embedded proteins "that are not so easily studied by the two main structural techniques, X-ray crystallography and NMR."
In a larger context, the new technology will aid "the rush" to transform genome sequencing projects' blueprints into broad views of protein function, says Mchaourab. "Central to this endeavor is structural biology that will transform these one-dimensional strings of DNA sequences into three-dimensional visual frameworks of how catalysis, ion conduction and energy transduction are carried out by proteins," he says. Structural biology and structural genomics are aimed at creating a catalog of the entire complement of unique proteins encoded by a genome.
Notes Mchaourab: "The ability of ACERT to transform this technology into a routine laboratory procedure will allow a whole new set of protein assays [testing and analysis] to emerge."
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