New method for studying protein structure could advance drug development
ARGONNE, Ill. (Dec. 10, 2004)—Structural changes in proteins can now be seen
in increased detail, using a new application of an existing technique. The
application, developed at the U.S. Department of Energy's Argonne National
Laboratory, could help produce lead drugs for disease therapy.
In research published in Chemistry
and Biology, the scientists report
the use of wide angle X-ray scattering (WAXS), an X-ray diffraction technique
that has previously been used to determine the crystalline structures of polymers.
The biologists adapted this materials science technique to study ligand-induced
structural changes in proteins. Ligands are molecules that can cause the
creation of complex compounds in protein structure.
The results Argonne scientists
achieved using WAXS are comparable to the already accepted predictions
of protein structures provided by X-ray crystallography, and are easier and
quicker to obtain. The results also show promise for using WAXS as a reliable
and high-speed tool for lead drug identification.
WAXS has the potential to identify medicinal drugs that can bind to target
proteins and to determine how effective drugs are at binding to and modifying
their targeted proteins. The technique is sensitive enough to tell the difference
between a ligand that's just sticking to the surface of a protein (a drug that
may have no effect) and a ligand that's actually changing the structure (a
drug that is more likely to be effective).
In the past, detecting this difference
required the use of several techniques combined. No other previous technique
has been able to distinguish the difference on its own, or as quickly.
“Wide angle X-ray scattering provides a real tool for identifying lead drugs,” said
co-author Lee Makowski of Argonne 's Biosciences
Division, “It will identify
a molecule that's good enough to be developed as a drug.”
The researchers believe WAXS will allow scientists to study more protein-ligand
interactions at a faster and cheaper rate than the existing laborious and expensive
X-ray crystallography.
“The data collection only takes a couple of minutes,” said Makowski, “So theoretically
an industrial pipeline could be set up that would only be limited by a few
minutes per protein-small molecule interaction.” Functional cell-based assays
(which are needed for other methods) currently take weeks, if not months to
complete — causing a bottleneck in data collection and analysis.
Furthermore, high quality crystal structures are tough to attain, and only
a limited number of proteins have documented crystal structures of the protein
with and without a ligand present.
“There is no other technique like this out there,” said co-author Diane Rodi
from Argonne 's Biosciences Division, “You can see more detailed changes that
take place in protein-ligand interactions in solution than you can with any
other technique. And more protein-ligand interactions can be tested.”
No previous available technique is able to show the magnitude of protein structure
change in the absence of a crystal structure. Small angle X-ray scattering
(SAXS) is able to show the size and shape of the protein, but does not show
details about the change. Circular dichroism spectroscopy (a method that provides
structural information on many types of biological macromolecules) doesn't
show the level of detailed changes WAXS provides.
WAXS does not require any crystallization, but uses the same X-ray scattering
procedure as crystallography. The technique involves placing the protein and
ligand in a water-based solution and then placing this solution in the path
of an X-ray beam. The resulting X-ray scattering pattern reveals information
about the detailed structure of the protein-ligand complex, which can then
be contrasted with a scattering pattern of the protein alone.
The researchers at Argonne tested four proteins plus and minus their corresponding
ligands using WAXS, which uses the intense X-ray beams at the BioCAT facility
in the Advanced Photon Source. The proteins were chosen based upon the best
structures available from the Protein Data Bank that had already been observed
with and without ligands using X-ray crystallography.
“We chose proteins that already had crystal structures so that we could assess
just how good the WAXS technique is,” said lead author Bob Fischetti, of both
Argonne 's Advanced Photon Source and Biosciences Division, “And of course
we wanted to convince people that what we were seeing is real.”
The tested proteins displayed changes that directly corresponded to those
documented from the crystal structures, proving the observations were real
and validating the method as a potential drug discovery tool.
The other author on the report, in addition to Fischetti, Rodi and Makowski
is David B. Gore (BioCAT, Advanced Photon Source, Argonne ).
The researchers have submitted a grant proposal request to the National Institutes
of Health for possible funding of future studies with WAXS. — Raquel
Harper
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For more information, please
contact Steve McGregor (630/252-5580 or media@anl.gov)
at Argonne.
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