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Long-Sought Protein Structure May Help Reveal How 'Gene Switch' Works
For Immediate Release: February 10, 2009
Michael E. Newman
The bacterium behind one of mankind’s deadliest scourges, tuberculosis, is helping researchers at the National Institute of Standards and Technology (NIST) and Brookhaven National Laboratory (BNL) move closer to answering the decades-old question of what controls the switching on and off of genes that carry out all of life’s functions.
In a new paper* the NIST/BNL team reported that it had defined—for the first time—the structure of a “metabolic switch” in its “off” state. The switch, which is found inside most types of bacteria, is in a protein called the cyclic AMP (cAMP) receptor (CRP). CRP is the binding site for cAMP, a small molecule that, once attached, serves as the signal to throw the switch. This “on” state of CRP then turns on the genes that help a microbe survive in a human host.
The researchers hope that once the switching mechanism is understood the data can be used to develop new methods for preventing tuberculosis and other pathogenic bacterial diseases. Additionally, they believe that learning how this specific protein switch works may provide insight into how genes in general are regulated.
The biochemical puzzle surrounding the CRP switch is the mechanism by which the protein binds cAMP at one end and attaches to—and activates—a gene at the other end. Believing that the protein somehow changes its overall shape after binding cAMP, researchers set out 25 years ago to study the structure of CRP in both its active state (with cAMP bound to it) and inactive state (without bound cAMP) to document where the morphing occurs.
Unfortunately, the task proved to be extremely difficult. Protein structures are worked out using a technique called X-ray diffraction, but it requires the protein to be crystallized, and proteins often are very hard to crystallize. Using CRP from the bacterium Escherichia coli, researchers were able to crystallize the protein in its active (“on”) state, but the structure of the inactive (“off”) E. coli CRP eluded them as attempts to crystallize it repeatedly failed. With only the structure of the “on” state defined, the genetic switching mechanism remained a mystery.
The breakthrough was achieved when NIST biochemist Travis Gallagher and colleagues substituted the CRP from Mycobacterium tuberculosis for the E. coli protein.
The team’s initial success—obtaining crystals of CRP in the “off” state—was dramatic given that no one had accomplished the feat in nearly three decades of trying with E. coli. But the real excitement came when the crystals were examined with X-ray diffraction.
“Although the M. tuberculosis protein in the ‘off’ state consists of two subunits that are genetically identical, we were surprised to see that the subunits were not structurally symmetrical as well,” Gallagher says. “In most two-subunit proteins, each subunit has the same conformation as the other.”
Gallagher says that the NIST/BNL team theorizes that it is the asymmetry in the absence of cAMP that prevents the protein from attaching to DNA. This, in turn, keeps CRP from activating genes when they are not needed.
The team’s next step is to crystallize M. tuberculosis CRP in the active state and define its structure, so that the both states of the protein from the same organism will be known.
* D.T. Gallagher, N. Smith, S-K Kim, H. Robinson and P.T. Reddy. Profound asymmetry in the structure of the cAMP-free cAMP receptor protein (CRP) from Mycobacterium tuberculosis. Journal of Biological Chemistry (published online Feb 4, 2009).