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unravel outer membrane transport mechanism
Yates, Life Sciences Editor
photo to enlarge
by L. Brian Stauffer
professor Emad Tajkhorshid
and graduate student James Gumbart used two software
programs developed in the U. of I.’s National
Institutes of Health Resource for Macromolecular
Modeling and Bioinformatics to painstakingly model
a critical part of a mechanism by which bacteria
take up large molecules.
CHAMPAIGN, Ill. — Using
X-ray data and advanced computer simulation and visualization software,
researchers at the University of Illinois have painstakingly modeled
a critical part of a mechanism by which bacteria take up large molecules.
Their findings provide a rare window on the complex interplay of proteins
involved in the active transport of materials across cell membranes.
Their study, which includes a collaborator from the University of Virginia,
appears online in the Biophysical Journal and was described in the May
25 edition of Science.
Transporting large molecules, such as vitamin B12, citric acid or other
vital nutrients across the outer membranes of Gram-negative bacteria
is not a simple task. The cells must be selective in what substances
they take up, and the outer membrane contains no energy-generating machinery
to power the job of hauling large molecules inside.
The new study examined an outer membrane transport system that depends
on an energy-generating inner membrane protein, TonB. This TonB-dependent
transporter (TBDT) contains a beta-barrel domain: a series of parallel
sheets that form a tunnel through which large molecules can pass. Another
region of the protein, the luminal domain, clogs this barrel until the
cell is ready to allow large molecules to pass through.
Crystallographic studies had shown that TonB binds to one end of the
luminal domain. Researchers had hypothesized that TonB somehow draws
the luminal domain out of the barrel or changes its conformation to
make way for the large molecules.
image to enlarge; click here to view results of the simulation.
of I. researchers simulated the interaction of part
of the inner membrane protein TonB (shown in red)
and the luminal domain (in green and dark blue) of
the outer membrane transporter. The yellow sphere
represents the point on the TonB protein at which
the force was applied.
had been inconclusive, however. Molecular biologists have difficulty
studying systems that involve complex interactions between proteins,
particularly when one domain moves into and out of a structure like
a beta-barrel, said biochemistry professor Emad Tajkhorshid, principal investigator on the study. “It’s
very difficult to assess this experimentally because they have to look
at the accessibility of certain (amino acids) before and after activation,”
Tajkhorshid said. “And sometimes the labeling agent that they
use diffuses into the beta-barrel and labels the inside: You don’t
know whether what you have labeled is happening inside or outside.”
To address these limitations, Tajkhorshid and graduate student James
Gumbart used two software programs developed in the U. of I.’s National Institutes of Health
Resource for Macromolecular Modeling and Bioinformatics. The programs,
NAMD and VMD, respectively, simulate and visualize complex molecular
interactions. By entering detailed data about the position and characteristics
of every atom in the system, the researchers ran simulations of various
scenarios to test which hypotheses were most feasible. Their work relied
on detailed crystallographic studies of the molecules provided by University
of Virginia researcher Michael C. Wiener.
“The good thing about simulations is that you can monitor the
position of every atom,” Tajkhorshid said.
The task was enormous, however.
“The fundamental motions of atoms that guide large conformational
changes happen on a very short time scale: femtoseconds,” Tajkhorshid
said. Each step relies on the completion of a previous step, so the
simulations take an extensive amount of time and considerable computational
The researchers addressed two key questions: First, could the bond between
TonB and the luminal domain withstand the force needed to pull the luminal
domain downward, away from the barrel? Second, how does the luminal
domain respond to force in order to expose a permeation pathway through
In the first simulation the researchers applied a force to TonB and
showed that the multiple hydrogen bonds between TonB and the luminal
domain were strong enough to remain intact while TonB pulled the end
of the luminal domain away from the barrel. The simulation also
showed the luminal domain gradually unfolding, changing its conformation
in ways that would open up enough space for a molecule of vitamin B12
to pass through the barrel. (Click on the link below the image of the
system to watch the results of the simulation.)
A second simulation exerted a force near the center of gravity of the
luminal domain, pulling it out of the barrel in one piece. This required
an enormous input of energy, however.
“The force that we applied was about 4,000 picoNewtons,”
Tajkhorshid said. “This is an order of magnitude higher than that
required to induce the unfolding. This clearly indicates that the unplugging
mechanism is very unlikely.”
Coupled with increasingly sophisticated crystallographic studies and
basic biological data, simulations help researchers study the interactions
of multiple proteins in complex systems, such as the TonB-dependent
transporter, Gumbart said.
“As we get more computational power, we’re looking less
at single proteins and more and more at the interaction of proteins,”
he said. “And this is going in the direction of what’s called
systems biology. This is essential because none of these proteins actually
works alone. Each works in concert with many other things.”
Tajkhorshid is also a professor of pharmacology in the College
of Medicine and an affiliate of the Beckman Institute and the Center
for Biophysics and Computational Biology in the College
of Liberal Arts and Sciences.
Funding for the research was provided by the National Institute of General
Medical Sciences at the National Institutes of Health.
Editor’s notes: To reach Emad Tajkhorshid,
call 217-244-6914; e-mail: email@example.com.
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