| Newswise
— 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.
Related Links:
Johns Hopkins Department of Chemical and Biomolecular
Engineering: http://www.jhu.edu/chbe/index.asp
Marc Ostermeier’s Web page: http://www.wse.jhu.edu/cheme/faculty/ostermeier/g
The Ostermeier Lab page: http://www.jhu.edu/~cheme/ostermeier
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