New, automated analytical and computational approaches and tools are needed to rapidly identify and characterize protein complexes. Michelle Buchanan and others have made ORNL a leader in "genomes to life" research involving characterization and imaging of these molecular machines.
The biology revolution launched by the Human Genome Program is not over yet, according to Michelle Buchanan, director of ORNL's Chemical Sciences Division and DOE's Center for Molecular and Cellular Systems. Knowing the sequence of the DNA bases in the human genome and the location of its genes is just a small step toward understanding life itself.
"Any protein encoded by a single gene is estimated to have up to 200 types of variations," Buchanan says. In a cell, a protein can be modified in different ways— by errors resulting from genetic mutations or post-translational modifications, for example. Proteins seldom act alone in cells but form complexes, which are the molecular machines of life.
"Protein complexes are the key to biological function," Buchanan says. "Our job is to identify normal and modified proteins; learn how protein complexes assemble; and determine the levels, cellular location, and dynamics of protein complexes. We must understand the network of reactions that occur in sufficient detail to predict, test, and comprehend the response of a biological system to changes."
ORNL, other Department of Energy national laboratories, and universities are using DOE funding to bring to the biology community the best information on protein complexes. Achieving this goal requires interdisciplinary teams of biologists, chemists, and computational scientists and the best available analytical and computational tools that DOE can offer.
DOE is interested in protein complexes because they are present in microorganisms related to the department's missions—energy production, environmental protection, and homeland security. The research is being conducted for DOE's Microbial Genome and Genomes to Life programs.
Buchanan is leading a multi-lab partnership (including four DOE labs and two universities) to study protein complexes in two types of bacteria, using several approaches. "Mass spectrometry will be a huge workhorse for protein complex identification," Buchanan says. "It is already being used for proteomics—identifying proteins and revealing their amino-acid sequences to get the parts lists— analogous to finding the order of DNA bases in gene sequencing."
Other tools will also be important for the pilot project. For example, optical microscopes will image proteins to which fluorescent tags have been attached. "We will watch protein complexes form and pull apart in real time," Buchanan says. Other useful techniques will be X-ray scattering, nuclear magnetic resonance spectroscopy, and neutron scattering. In 2005 ORNL's High Flux Isotope Reactor will have the world's best small-angle neutron-scattering facility, the Center for Structural Molecular Biology, which is funded by DOE's Office of Biological and Environmental Research.
As an energy research agency, DOE focuses on microbes that produce clean fuels, such as methane, methanol, and hydrogen. Buchanan's project is studying Rhodopseudomonas palustris, whose talents include hydrogen production. Another microbe being studied is Shewanella oneidensis, which can turn soluble metallic compounds in soil and water into insoluble compounds. Scientists hope that soluble uranium can be converted into insoluble uranium on DOE sites so it will stay in the soil or sink into sediments, rather than dissolve in water where it could be transported off site. Because of its environmental restoration mission, DOE is interested in this microbe and other microbes that convert organic pollutants into less toxic materials.
The project partners are working to improve technologies for fishing protein complexes out of bacteria using affinity tags and reagents and isolating the complexes for analysis by mass spectrometry. ORNL's "lab on a chip" technology might make this pipeline more efficient. The partners are also improving high-throughput computational tools for rapidly analyzing, interpreting, and communicating on web sites the volumes of data on, for example, five novel proteins that the partners discovered during research on R. palustris.
One long-term objective is to provide high-quality data on protein complexes to the biological community, much like the data large sequencing centers provided during the Human Genome Program. Meeting this goal will require developing whole new technologies, as well as improving and automating current state-of-the-art but labor-intensive technologies.
"This project is an extreme challenge for analytical chemists and computational scientists," Buchanan says. "But it will be a definite boon for biologists."
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