Scientists at the U.S. Department
of Energy's (DOE) Lawrence Berkeley National Laboratory have developed a
fast and efficient way to determine the structure of proteins, shortening a
process that often takes years into a matter of days.
Greg Hura at the Sibyls beamline at the Advanced Light Source at Berkeley Lab. The beamline has two interchangeable end stations, one for macromolecular crystallography and one for small angle X-ray scattering (SAXS). Photo: Lawrence Berkeley National Lab - Roy Kaltschmidt, photographer
The high-throughput protein pipeline could allow scientists to expedite the
development of biofuels, decipher how extremophiles thrive in conditions that
kill most organisms, and better understand how proteins carry out life's
vital functions.
The technique will help scientists keep pace with the growing flood of data
stemming from genomic studies of organisms and environmental samples such as
seawater and soil. Every new gene identified in these studies codes for a protein,
and the structure of each protein must be characterized in order to determine
what it does. Current structural characterization techniques are slow, however,
meaning newly discovered proteins and their many complexes keep piling up, their
function remaining a mystery.
“There's a bottleneck in structural genomics, and our system addresses
that,” says Greg Hura, a scientist in Berkeley Lab's Physical Biosciences
Division. He developed the technique with John Tainer of Berkeley Lab's
Life Sciences Division and the Scripps Research Institute in La Jolla, CA. Michael
Adams and other scientists from the University of Georgia also contributed to
the research.
Their work is published in the July 20 online edition of the journal Nature
Methods.
The team developed the protein pipeline at the Advanced Light Source (ALS),
a national user facility located at Berkeley Lab that generates intense light
for scientific research. At a beamline called SIBYLS, they used a technique
called small angle x-ray scattering (SAXS), which can image a protein in its
natural state, such as in a solution, and at a spatial resolution of about 10
angstroms, which is small enough to determine a protein's three-dimensional
shape. The brilliant light generated by the Advanced Light Source minimizes
the amount of material required for each experiment, which makes the technique
practical for almost any biomolecule.
To maximize speed, Hura installed a robot that automatically pipettes protein
samples into position so they can be analyzed by x-ray scattering. And to analyze
the resulting data, they used the supercomputing resources of the U.S. Department
of Energy's National Energy Research Scientific Computing Center (NERSC), which
is based at Berkeley Lab. The supercomputer's clusters can churn through data
for 20 proteins per week, or more than 1000 macromolecules per year.
The result is a system that moves at breakneck speed compared to current techniques
used to determine the shape and structure of proteins: x-ray crystallography
and nuclear magnetic resonance. Recently, in the span of one month, the team
used the system to resolve the structure of 40 proteins from Pyrococcus furiosus,
a microscopic extremophile that can live at 100°C.
“This would have taken several years with x-ray crystallography,”
says Hura. “What used to take years, now can takes weeks.”
Adds Tainer, “We can now obtain structural information in solution on
most samples, rather than the 15 percent obtained by the best of the current
Structural Genomics Initiative efforts employing nuclear magnetic resonance
and crystallography. “
The Berkeley Lab team chose P. furiosus because it is an intriguing candidate
for the production of clean energy and other applications. It has a pathway
that produces hydrogen, which is a potential alternative fuel. And many industrial
processes are highly acidic and very hot — conditions that P. furiosus
loves.
“If we could learn which of the organism's proteins allow it to thrive
in these conditions, then maybe we can apply them to energy production and other
applications,” says Hura.
Future synthetic biology efforts may involve taking a useful protein or a network
of proteins from one microbe, and importing it into another microbe. In order
to do this, scientists must learn how much of the network needs to be imported
and still have it be able to do its job. This requires analyzing individual
proteins in hundreds of different conditions.
“This is where our system will have a big impact. We can do this type
of structural analysis in a matter of weeks, as opposed to years with crystallography,”
says Hura.
Of course, such speed doesn't come without tradeoffs. X-ray crystallography
yields extremely high-resolution images, while small angle x-ray scattering
yields a protein's shape at a much lower resolution of about 10 angstroms
(one angstrom is one ten-millionth of a millimeter).
But the level of information offered by x-ray crystallography isn't always
necessary. Sometimes, simply knowing if one protein is similar in shape to another
is enough to learn its function. And SAXS makes up for what it lacks in precision
by providing accurate information on the shape, assembly, and conformational
changes of proteins in solution.
“We can have less information and still answer the questions that need
to be answered,” says Hura, adding that their technique will help usher
in the next phase of genomics research. “The number of genes being identified
is growing at a huge rate. We need to keep pace with this and learn about all
the proteins encoded in these genes.”
Adds Tainer, “This pipeline is an example of the stunning impact we can
achieve by combining physics and engineering with structural biology, which
is possible at government labs like Berkeley Lab.”
The multidisciplinary work, which was conducted at Berkeley Lab's Advanced
Light Source at beamline 12.3.1, also known as SIBYLS (Structurally Integrated
BiologY for Life Sciences), relied on resources provided by three separate offices
within the DOE Office of Science (SC). This work itself was supported in part
by SC's Office of Biological and Environmental Research (BER). The ALS
is supported by SC's Office of Basic Energy Sciences, while the beamline
is supported in part by BER. NERSC is funded by SC's Office of Advanced
Scientific Computing.
To aid communication of results, the team created a web-accessible database,
www.Bioisis.net, which archives all experimental details associated with each
analyzed sample.
“Robust, high-throughput solution structural analyses by small angle
X-ray scattering (SAXS)” by Greg Hura, Angeli Menon, Michal Hammel, Robert
P. Rambo, Farris Poole, Susan Tsutakawa, Francis Jenney, Scott Classen, Kenneth
Frankel, Robert Hopkins, Sung-jae Yang, Joseph Scott, Bret Dillard, Michael
Adams, and John Tainer is published online July 20 in the journal Nature Methods.