Improving significantly on an early prototype, Johns
Hopkins 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 Whiting School's
Department of
Chemical and Biomolecular Engineering. "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. Johns
Hopkins has applied for a patent covering the molecular
switch and methods of producing it.