Improving significantly on an early prototype, Johns Hopkins University researchers have found a new way to join two unrelated proteins to create a molecular switch, a nanoscale "device" in which one biochemical partner controls the activity of the other.
Lab experiments have demonstrated that the new switch performs 10 times more effectively than the early model and that its "on-off" effect is repeatable.
The new technique to produce the molecular switch and related experimental results are reported in the November issue of the journal Chemistry & Biology. The paper builds on earlier research, led by Marc Ostermeier, which demonstrated that it was possible to create a fused protein in which one component sends instructions to the other. The second then carries out the task.
"Last year, we reported that we'd used protein engineering techniques to make a molecular switch, putting together two proteins that normally had nothing to do with one another, but the switching properties of that version were insufficient for many applications," said Ostermeier, an assistant professor in the Department of Chemical and Biomolecular Engineering at Johns Hopkins. "With the new technique, we've produced a molecular switch that's over 10 times more effective. When we introduce this switch into bacteria, it transforms them into a working sensor."
As in their earlier experiments, Ostermeier's team made a molecular switch by joining two proteins that typically do not interact: beta-lactamase and the maltose binding protein found in a harmless form of E. coli bacteria. Each of these proteins has a distinct activity that makes it easy to monitor. Beta-lactamase is an enzyme that can disable and degrade penicillin-like antibiotics. Maltose binding protein binds to a type of sugar called maltose that E. coli cells can use as food.
In the previous experiments, the researchers used a cut-and-paste process to insert the beta-lactamase protein into a variety of locations on the maltose binding protein, both proteins being long chains of amino acids that can be thought of as long ribbons. In the new process, the team joined the two natural ends of the beta-lactamase chain to create one continuous molecular loop. Then, they snipped this "ribbon" at random points before inserting the beta-lactamase in random locations in the maltose binding protein. This technique, called random circular permutation, increases the likelihood that the two proteins will be fused in a manner in which they can communicate with each other, Ostermeier said. As a result, it's more likely that a strong signal will be transmitted from one partner to the other in some of the combined proteins.
In their new paper, the Johns Hopkins team reported that this technique yielded approximately 27,000 variations of the fused proteins. Among these, they isolated one molecular switch, in which the presence of maltose, detected by one partner, caused the other partner to increase its attack on an antibiotic 25-fold. They also showed that the switch could be turned off: When the maltose triggering agent was removed, the degradation of the antibiotic instantly slowed to its original pace.
Ostermeier believes the same molecular switch technology could be used to produce "smart" materials, medical devices that can detect cancer cells and release drugs, and sensors that could sound an alarm in the presence of chemical or biological agents. His team is now seeking to create a molecular switch that fluorescently lights up only in the presence of certain cellular activity. "We've proven that we can make effective molecular switches," he said. "Now, we want to use this idea to create more interesting and more useful devices."
Gurkan Guntas, a doctoral student in Ostermeier's lab, was lead author on the new Chemistry & Biology paper. The co-authors were Ostermeier and Sarah F. Mitchell, a doctoral student in the Program in Molecular Biophysics at Johns Hopkins. The research was supported by a grant from the National Institutes of Health. The Johns Hopkins University has applied for a patent covering the molecular switch and methods of producing it.