New Technique Enables Studying Proteins In Living Cells
Duke biochemists have reported a 3-dimensional nuclear magnetic resonance (NMR) spectroscopy technique that enables them to deduce the structural characteristics of a protein inside a living cell. The achievement is significant because it brings together the important areas of biochemistry and cell biology. While other structural analytical techniques typically involve isolating proteins in test tubes or crystallizing them to determine their structures, the NMR spectroscopy method enables detailed analysis of the protein in its natural biological environment, said the biochemists.
Conventional protein analytical techniques have proven powerful tools for studying proteins, but they cannot capture the multiple subtle -- and often important -- interactions that proteins experience in a living cell, said the biochemists. These interactions can profoundly influence their conformation inside cells and their biological function, they said.
The researchers said their "multidimensional" NMR technique could have broad application in basic understanding of proteins and in drug development. It could enable scientists to explore protein function in detail in living cells and to eavesdrop on the action of experimental drugs in affecting protein function.
The paper on "Multidimensional NMR Spectroscopy for Protein Characterization and Assignment Inside Cells" -- by graduate student Patrick Reardon and Professor Leonard Spicer -- was published online July 13, 2005, by the Journal of the American Chemical Society. Spicer is a professor of radiology and biochemistry and director of the Duke University NMR Center . The state-of-the-art NMR instrumentation used in the research was funded by the National Institutes of Health and the National Science Foundation.
Magnetic Resonance spectroscopy involves the use of powerful magnetic fields and radio waves to probe the structure and dynamics of molecules by measuring the fingerprint of individual nuclei of the atoms that constitute target molecules. Long a powerful technique for analyzing molecular structure of organic molecules, biochemists have been extending NMR spectroscopy to probe the structure of larger and more complex macromolecules such as proteins. Discovering the structure of proteins is critical to biology, since they are the "workhorse" molecules of the cell and often catalyze or regulate the machinery of life.
"There have been a great many questions about whether proteins, particularly functional proteins, behave the same way in cells as they behave in vitro and in isolated systems," said Spicer. "And until now, structural biologists have had to isolate and purify proteins for study. While this has yielded invaluable information, what we may miss are the cooperative effects of proteins in living cells -- for example, when they assembly into macromolecular machines in the cell."
In their new technique, which incorporates "fast 3D NMR" methods the researchers used cryogenically cooled NMR probes to enhance sensitivity and rapidly obtain a series of characteristic signals from a protein in a living cell. . They then used a data processing approach currently being developed by their colleagues Professor Pei Zhou, Dr. Ron Venters and graduate student Brian Coggins called "projection reconstruction," to represent the signature "fingerprint" of the protein as it exists in a living cell. Previously, conventional methods took days to collect the required data in 3 dimensions, which rendered the detailed analysis of proteins in cells all but impossible. However, in their paper, Reardon and Spicer described an approach that reduced to hours the time to acquire sufficient data.
In their experiments, the researchers used as a test molecule, a protein called GB-1 and produced it in the common gut bacterium E. coli. To render the protein "visible" to NMR, they labeled the molecule with stable heavy isotopes of nitrogen and carbon. And to ensure that the proteins were produced in sufficient concentrations to be detectable using NMR, they engineered the bacterium to overproduce the protein.
"Our colleagues are very excited by this technique, because it represents a spectroscopic approach that enables them to better understand the behavior of a protein in vivo," said Spicer.
In further studies, Spicer and his colleagues will progress to studying proteins in living yeast cells, since yeast is a more advanced "eukaryotic" cell that is closely related to animal cells. In such cells, proteins can undergo important structural alterations after they are synthesized that do not occur in bacteria. Thus, said Spicer, studies of proteins in yeast can be more directly applicable to understanding human proteins and their interactions.
Also, he said, researchers can use the technique to understand how multiple target proteins interact in living cells by over producing them together in bacterial or yeast cells and performing NMR spectroscopic analyses. Such interactions among proteins are central to the formation of the protein complexes that carry out such functions as repairing DNA damaged by radiation. They also are associated with processes leading to plaque formation in neurodegenerative diseases.
Multidimensional NMR spectroscopic characterization and the analyses of protein interactions within cells could be invaluable to pharmaceutical companies in developing new drugs, said Spicer.
"It should be possible to deliver small molecules that inhibit or promote activity into such cells and to follow their interaction with protein enzymes in vivo," said Spicer. "Then you can see what actually happens. You can see how the protein was modified during the binding and inhibition process and measure the output of an enzyme after the fact. Such a capability could offer an enormous advantage in understanding that interaction and in enhancing it to refine the structure of a candidate drug molecule," he said.