Protein Structures Solved Faster with More Detail than Ever

As scientists across the country finished “mapping” the human genome, Argonne researchers were already fine-tuning the technology to meet the next hurdle—slashing the time needed to determine the genome’s three-dimensional structure.

The key to Argonne’s speedy technique is the Structural Biology Center (SBC) at the Advanced Photon Source (APS). The APS produces the country’s most brilliant X-ray research beams—allowing rapid data collection and structure determination at atomic-scale resolutions. In 1999, the SBC solved more than 100 protein structures.

“Being able to read a genome is important,”explained Andrzej Joachimiak, SBC director, “but the structure is responsible for the function. Now that we know the proteins from the genome, we can determine their structures and functions to realize the full potential of the genetic information.”

Argonne provides the highest-resolution protein structures available anywhere in the world and does it with great speed. Solving a protein’s structure used to take months or years; with new tools and techniques developed at Argonne, it now takes only hours or days. Argonne researchers have also automated gene cloning and expression, protein production and crystallization, and are using sophisticated computer techniques to simplify structure solving.

The genome is the set of genetic instructions in the cells of all living things. Those instructions are written in the sequence of bases—adenine (A), guanine (G), cytosine (C) and thymine (T)—that form the two strands of DNA’s double helix. Sequences of these bases create specific amino acids, which link in different combinations to make different proteins. These proteins are responsible for all the genetic traits any living organism carries.

When scientists mapped the human genome, the result was a string of As, Gs, Cs and Ts. With 100,000 genes in the human genome, the task was immense. The human genome is only the latest addition to the catalog of more than 40 known genomes. Now researchers need to understand the structure and function of each protein, because this knowledge will ultimately help doctors treat, cure or prevent disease.

To determine a protein’s structure, the brilliant APS X-rays are shined on tiny crystals of the protein at liquid nitrogen temperatures. The pattern of X-rays produced by the sample is captured in a kind of digital camera, called a “CCD detector,” and the data are mathematically converted to a three-dimensional image.

For example, the crystal structure of immune cells called CD4 T cells was determined in a few hours. Data collection took less than one hour. The structure provides clues to how the immune system identifies enemy threats and is helping researchers to understand the human response system . Results of this research were published in Science.

Researchers also determined the structure of a large detoxifying enzyme, cyanase, which helps certain bacteria to regulate carbon dioxide levels and to neutralize toxic chemicals. It may be important for technologies that use biological species to clean the environment. The data quality was so high that researchers automatically produced the protein structure in a few days. It was published in Structure magazine. Other structures studied at the SBC may provide insights into diabetes, muscular dystrophy and high blood pressure.

SEEING IS UNDERSTANDING
Seeing a structure helps researchers understand a molecule’s function. Knowing a protein’s structure is valuable in drug design because pharmaceutical companies can develop drugs to hook tightly to it. Such medications can be more effective and provide fewer side effects.

Some Argonne biologists and computer scientists are focusing on finding potential targets for antibiotics. Biologists identify genes and metabolic pathways that are unique to disease-causing organisms so that they may be used to fight the disease without harming the human.

The Computational Biology Group developed WIT3, an interactive database that stores genomic and metabolic information and provides tools for users to access the data and construct their own models of the sequences of As, Cs, Gs and Ts.

The researchers originally developed a computer program called WIT, or What Is There, to store and compare genomic information on the World Wide Web. The new WIT3 is automated, speeding the process of retrieving data and constructing models. WIT3 is available to researchers on the World Wide Web. The site has about 20,000 users and receives between 3,000 and 5,000 visits a day.

The success of Argonne’s structural genomics program depends on the technological advances of synchrotron facilities such as the APS, molecular biology and crystallography methods, robotics, and computer hardware and software. But the process begins with two time-consuming, vital tasks in a more traditional laboratory.

HENRY FORD MEETS BIOLOGY
First biologists clone a protein by snipping pieces out of its genome and placing them in “expression bacteria,” which make many protein copies. Argonne researchers created a modern production line using robots to automate this time-consuming cloning task. The Robotic Molecular Biology Facility can produce 400 to 800 protein clones each week; manual methods produce only 20 to 40. Then biologists coax these purified proteins to form crystals for X-ray crystallography in the Structural Biology Center.

THE FUTURE
Argonne researchers continue to refine the structural genomic process to make it even faster. For example, the robotic lab plans to quadruple production in 2001 by increasing from 96 to 384 the number of miniature test- tube-like wells the robot can handle at a time. Also, the SBC will take advantage of the APS’s brighter X-rays using new larger, faster X-ray detectors that provide information in greater detail.

In the future Argonne may be able to offer a structural genomics assembly line to many users, explained Joachimiak. “Researchers can send us their crystals to be placed in the beamline using robots and have the data remotely collected and processed in ‘real time.’”

For more information please contact Evelyn Brown at 630-252-5510

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