New crystallization method to ease study of protein structures
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ARGONNE, Ill. (March 7, 2007) – Researchers at the Midwest
Center for Structural Genomics (MCSG), the Structural
Genomics Consortium (SGC) and the Structural
Biology Center (SBC) at the U.S. Department of Energy's Argonne National
Laboratory have developed a new technique for crystallizing proteins that will
ease experimentation into protein structures.
In order to study protein structures, biologists must turn what is essentially
a soup of purified protein into crystals that have a consistent and ordered
structure. Each protein consists of a chain of amino acid subunits that twists
into helices, ribbons and loops. Some proteins have less tidy molecular structures
than others; in these, disordered amino acid chains dangle off the protein
like split ends.
Crystallizing proteins that contain many of these flexible regions takes much
more work and patience than working with more organized ones, said Argonne
senior biologist Andrzej Joachimiak, who led the Argonne research effort. "We've
tried to find a way to remove the disordered parts using computer modeling,
but that's been a challenging process," he said. "This new experimental
method is fast, inexpensive and can be applied to many different targets, from
bacterial pathogens to human proteins."
In order to try to boost the efficiency of the crystallization process, Joachimiak
and his colleagues at the MCSG and SGC inserted a protease—a certain type of
enzyme that breaks down the bonds that connect a protein's amino acids.
Once added, the protease preferentially bound to the proteins at the disordered
regions, snipping off the loose ends like a molecular barber. The researchers
successfully crystallized and examined nine of these newly shorn proteins that
previously had resisted attempts to study them using X-ray crystallography.
"This simple technique offers an opportunity to uncover and characterize
the structures of dozens of proteins that up until now we had to study using
much more laborious and expensive approaches," Joachimiak said.
This process, known as "limited in situ proteolysis," represents
one of several potential "salvage pathways" that biophysicists could
use to create more usable protein crystals and reduce waste, Joachimiak said.
Currently, scientists' efforts to manufacture and then study a workable crystal
on Argonne's Advanced Photon Source yield structural data only about 15 percent
of the time. By using proteases to digest part of the protein sample, the Argonne
scientists achieved a six percent boost in efficiency.
Joachimiak cautioned that scientists do not have a way to successfully crystallize
every protein, even with the use of proteolysis. "There will still
be some that are resistant," he admitted, "but we are making enormous
strides in our understanding of how exactly these essential substances work."
A research paper, "In
situ proteolysis for protein crystallization and structure determination," that detailed the study appeared in the
December 4 issue of Nature
Methods. The study's X-ray data were collected at the SBC
beamlines at the Advanced Photon Source. The MCSG and SGC represent a collaboration
of Argonne scientists as well as scientists from Canada and Europe.
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of Science.
By Jared Sagoff.
For more information, please contact Angela Hardin
(630/252-5501 or ahardin@anl.gov)
at Argonne.
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