This knotted protein comes from a microorganism called methanobacterium thermoautotrophicum that breaks down waste products and produces methane gas.

Automating structural biology expedites biologists’understanding of protein functions

The race to determine the three-dimensional structure of the proteins of the human genome is on, and Argonne’s Structural Biology Center (SBC) researchers are at the front of the pack in developing new tools and techniques to speed the process, as well as determining a record number of structures.

“The protein structure is the key,” Structural Biology Center Director Andrzej Joachimiak said, “to using the information to cure, treat and prevent the disease, because it is the structure that is responsible for the protein’s function.” Proteins govern the body’s activities, from creating enzymes that digest food to carrying oxygen throughout the body.

Biologists are fine-tuning the high-tech production lines they have developed to condense time-consuming wet-lab processes. Simultaneously, crystallographers are speeding the process of discovering three-dimensional structures using X-rays and computers.

As lead laboratory of the National Institute of General Medical Sciences’ Midwest Consortium for Structural Genomics, it is Argonne’s job to morph the labor-intensive task of creating crystals into a rapid, precise production line. The goals are to cut the average cost of analyzing a protein from $200,000 to $20,000 and to slash the average time from months to hours and days. Argonne’s pipeline has already reduced the cost of solving a protein crystal to $40,000.

The protein structure determination pipeline
To achieve this goal, researchers are building a crystallization pipeline using multiple robots to process several proteins simultaneously. In fiscal year 2002, the center made 100 crystals for research; in 2003, 150. The goal for 2004 is 250 crystals.

“Our biggest bottleneck is crystallization,” explained Joachimiak. “The human genome project gave us so much to study that we must have a faster way to use that information to understand the proteome’s structure.”

Robots perform the repetitive, time-consuming wet-lab process. The process begins with a robot cloning a gene of the protein by snipping pieces of the genome and placing them into specially designed bacteria to produce thousands of protein copies.

Another robot purifies the proteins. This previously took two to three days per protein. Argonne researchers are now purifying six a day with a goal to produce several thousand a year.

There are many more steps in the process, and Argonne researchers work with suppliers to create robots for each step.

Crystal light
Once a protein has been crystallized, it is X-rayed at the Advanced Photon Source, this hemisphere’s most efficient source of X-rays. Argonne’s SBC, funded by the Department of Energy’s Office of Biological and Environmental Research, operates the fastest beamline of its type in the world.

Computers process the multiple sets of X-ray data and turn them into a three-dimensional protein image using newly developed semi-automated procedures. Visualizing the protein provides researchers with clues to how it works. If the protein is related to a disease, researchers use rational drug design that allows them to design specific molecules to block the protein’s natural interactions. Traditional drug design requires random testing of thousands of compounds to do the same thing.

Deposits in the bank
After the structures are solved, they are deposited in the Protein Data Bank for other researchers to study. Argonne’s SBC beamlines are the world’s most productive. In fiscal year 2003, SBC researchers submitted a record 150 structures to the Protein Data Bank; 45 of those were produced by the Midwest consortium.

“In the first seven months of 2003,” Joachimiak said, “25 protein structures were solved at the SBC. When you consider that it took seven years to determine the first 25 structures, you see how amazing the new processes are.”

SBC researchers are setting other records, too. Macromolecular crystallographer Rong-guang Zhang set a record for the fastest time ever for depositing a structure in the Protein Data Bank. From crystal in the beamline to Protein Data Bank took him less than 30 hours. Traditionally it has taken weeks and months.

Biologists using the SBC are also making other interesting discoveries, such as a protein with a surprising feature: a knot, which was found by Youngchang Kim. This is the first time a knot has been found in a protein from the most ancient type of single-celled organism, an archaebacterium, and only the second time a knot has been seen in any protein structure.

“Protein folding theory previously held that forming a knot was beyond the ability of a protein,” Joachimiak explained. He suggested that the newly discovered knot stabilizes the amino acid subunits of the protein. “Nature must really want to secure this structure to go to the trouble of tying a knot,” Joachimiak said.

Biologists are also investigating unknown territory. Little is known about half of the proteins in the human genome. Traditional methods give no clues to structure or function. Researchers are breaking ground by determining the structures of what biologists call “hypothetical” proteins. These are proteins that are distinct from any known structures, but are considered important because they exist in several branches of life. No longer “hypothetical,” these structures are added to the Protein Data Bank, awaiting findings of similar protein structures to provide clues to their role in life.

See www.sbc.anl.gov

For more information, please contact Evelyn Brown.

Next: SARS main protease structure determined at APS

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