August 26, 2005
Inteins aren't what you'd call "helping enzymes." In other words, they
don't assist other proteins in the reactions that transform them from
their primary states into the protein complexes that enable them to
perform their unique biological functions. Instead, they remove
themselves entirely from the proteins of which they are components--and
then splice the remaining parts together to form a whole molecule.
"You can think of a protein as a kind of ribbon," says Phil Shemella, a
graduate student in the department of physics at Rensselaer Polytechnic
Institute. "At two locations, the ribbon is cut, and the middle--the
intein--falls away." These inteins are autocatalytic--they already
contain the enzyme necessary to accomplish the cleaving and splicing
that forms the new protein structure.
"We sometimes call them 'selfish proteins,' or 'selfish DNA,'" says
Saroj Nayak, an assistant professor of physics at RPI. Nayak is
Shemella's advisor and a principal investigator in a multidiscplinary
investigation into why inteins behave the way they do. "Inteins are by
themselves--they don't care about the rest of the physical area, they
don't need anything else, they're entirely self-contained."
Splicing and cleaving
Inteins have been observed in some 100 proteins found in around 30
single-celled organisms, including some yeast, bacteria, some
thermophilic microorganisms often found around deep-sea vents, and
human pathogens--including the bacterium that causes tuberculosis. In
fact, current knowledge about inteins has already been exploited in
researching new antibiotic treatments for the widespread communicable
disease. Nayak, Shemella, and their collaborators at RPI and the New
York State Department of Health's Wadsworth Center are working toward
the creation of a nanoswitch that would be useful for turning
biological processes on and off.
However, not much is really known about the exact mechanism by which
the intein excises itself--a process researchers refer to as "splicing
and cleaving." It is known that the intein breaks the protein up into
groups of around fifteen amino acids each. But which amino acids are
responsible for splicing the protein remnants back together is unclear.
"There are a lot of arguments about why certain amino acids are shared
at certain locations for proteins to have the splicing behavior," says
Shemella, whose own work focuses on examining one specific area of one
particular amino acid grouping, or motif. "From the literature--that's
the best understanding we have of the mechanism--we know that the
conserved residues must be important for cleaving. We know that we can
alter three amino acids and prevent the cutting from occurring on the
left-hand side, and we know that the process speeds up at lower pH
levels, but we don't know why."
To answer this question, Nayak, Shemella, and their collaborators,
Georges Belfort and Shekhar Garde of the chemical and biological
engineering department at RPI and Marlene Belfort and Vicky Derbyshire,
molecular biologists at the Wadsworth Center, are using molecular scale
modeling on NCSA's Tungsten to simulate intein behavior and the
environment that would trigger splicing and cleaving. Their quantum
mechanical/ molecular mechanical (QMMM) method uses the Gaussian03 code
to combine quantum mechanics with classical molecular dynamics; the
quantum mechanics component allows them to begin the simulation ab
initio, without requiring experimental parameters.
Inside the protein molecule
During the simulation, Nayak and Shemella run calculations on the
entire molecule and the solution surrounding it (water) using classical
molecular dynamics methods, while they run calculations on the
reactions within the molecule itself using quantum mechanical methods.
It's important for studying this reaction, because at the same time
that the dynamics is going on there's a chemical reaction going on, so
you have to have both quantum mechanics and molecular mechanics,"
explains Nayak. "Ideally, you should use quantum mechanics for the
complete system, but practically that's not possible, because the
system's too large. But you need the system to be too large, because
you want to give the proteins all the freedom that they need, and you
want to mimic reality as much as possible." The size of a typical run,
according to Nayak, is around 6,000 to 7,000 atoms.
Gaussian03 runs on anywhere from one to eight processors at a time for
a total of around 40 hours, and Shemella and Nayak have been pleased
with the overall performance. "Tungten has been pretty nice, pretty
fast," Shemella says. "It's well-maintained, too." To ensure that their
simulations run smoothly, they have been in close contact with Dodi
Heryadi of NCSA's User Support and Consulting Group. "NCSA does a
really excellent job in supporting whatever we need," says Nayak. "It's
really remarkable that they take the time to get the software, to help
us set up. I don't think it would have been possible to do this in our
home institution--the infrastructure and the time it would have taken
just would not have been possible at our university, or at many
Using a snapshot of the protein structures from the molecular dynamics
simulation performed by Garde's group, the collaborators ascertain what
the possible key aspects (for example, temperature, acidity, etc.)
might be that trigger the intein's mechanism, alter them, and run more
simulations accordingly. They have narrowed the field down to five
possible mechanisms. "That's where the fun begins," says Nayak. "We can
take our results to the experimentalists [Belfort at RPI and Belfort
and Derbyshire at Wadworth], and they can do experiments involving the
mutations we've predicted. And if we have a two-way cross-check, you
have more confidence of things going the way they're supposed to."
The multidisciplinary nature of the collaboration to investigate intein
mechanisms brings a wide variety of scientific goals into
synchronicity. The combined goal, from the standpoint of molecular
biology, chemical engineering, and physics, is to exploit the intein to
create molecular nanoswitches that respond to stimuli such as light and
heat for use in biotechnology. But Shemella identifies an even more
basic goal. "Any time you can figure out what makes a mechanism work,
why it works, how it works -- that's important."