June 26, 2006
Nanotechnology Drives Protein Engineering, New Approach to Drug Discovery
Using a nanoscale spring built from a molecule of DNA, investigators from the University of California, Los Angeles, have taken a significant step toward a new approach to protein engineering. This new tack to modifying protein function, note the researchers, could lead to novel ways of killing cancer cells.
Reporting their work in the Journal of the American Chemical Society, Giovanni Zocchi, Ph.D., and graduate student Brian Choi show that they can use a nanoscale spring to control the function of an enzyme complex known as protein kinase A (PKA), which plays a fundamental role in the cell's signaling and metabolic pathways. Normally, PKA activity is controlled in the cell by a ubiquitous messenger molecule called cyclic AMP (cAMP), but the researchers demonstrated that they could control this enzyme’s activity mechanically with a nanodevice that the researchers attached to the enzyme complex. The nanodevice is essentially a molecular spring made of DNA.
"Molecular biologists have been trained for 50 years to think that because the sequence of amino acids determines a protein's structure and the structure determines its function, if you want to change the structure, the way to do so is to change the sequence of amino acids,” said Zocchi. “While that approach is correct, it is not the only way. We are introducing the notion that you can keep the sequence but change the structure with mechanical forces.”
PKA, a complex of four protein molecules, contains two regulatory subunits and two catalytic subunits. The investigators mechanically activated PKA by using a piece of DNA that places a controlled mechanical stress on two specific points in the regulatory subunit. This mechanical stress causes that subunit to fall off from the catalytic subunit, activating the enzyme. To obtain the desired effect, the mechanical tension is applied at specific locations in the regulatory subunit. Knowing those locations requires a detailed understanding of the structure of the enzyme.
Proteins are switched on and off in living cells by a mechanism called allosteric control. In the allosteric control process, proteins are regulated by other molecules that bind to their surface, inducing a change of conformation, or distortion in the shape, of the protein, which in turn can either activate or inactivate the protein.
Cyclic AMP binds to PKA's regulatory subunit and induces a change of conformation that leads to the catalytic subunit's detaching from the regulatory subunit; this separation of the two subunits is how the enzyme complex is turned on in the cell. "We can activate the enzyme mechanically, while leaving intact the natural activation mechanism by cyclic AMP," said Zocchi. "We believe this approach to protein control can be applied to virtually any protein or protein complex."
Going forward, Zocchi says he wants to determine if he can make similar molecular springs that kill a cell based on the genetic signature of the cell. "Cancer cells would be an obvious application,” said Zocchi. “This will however require many further steps. So far, we have only worked in vitro. The exciting part is, from the outside, cancer cells can look like normal cells, but inside they carry a genetic mark.” Indeed, experiments using versions of PKA with slightly different structures suggested that it would be possible to alter the activity of key proteins that differ between healthy and malignant cells.
This work is detailed in a paper titled, “Mimicking cAMP-dependent allosteric control of protein kinase A through mechanical tension.” This paper was published online in advance of print publication. An abstract is available at the journal’s website.