by Laura Buenning
Proteins are life’s worker-bee molecules. They do what’s needed to keep the cells alive, playing a crucial role in the structure, function, and regulation of living organisms.
Often large and complex, life-sustaining molecules are composed of multiple stable units called domains—each with a distinct structure and function; each conserved in near identical form from specie to specie. Understanding how they work involves identifying the domains, figuring out what they do, and determining how they fit together—a difficult proposition given the millions of possible arrangements, says JDRD team leader Eric Boder.
Boder, graduate student Maryam Raeeszadeh-Sarmazdeh, and an LDRD team led by Hugh O’Neill are tracking down the molecular configuration of the cellulose synthase (CesA) proteins responsible for synthesizing the fibrous cellulose material found in a plant’s cell wall.
CesA proteins have lots of stable domains, which have to assemble into a particular structure for the protein to work properly. Boder says, “X-ray crystallography and other techniques can tell us where the atoms are in the domains, but we still don’t know how all the pieces fit together; so you can’t really say how the whole thing works.”
Their goal is to build a tool kit for identifying the structure of extremely complex proteins. O’Neill’s team will combine data from crystallography and a neutron scattering method to model CesA in action. Both teams will expand the use of a clever technique Boder invented in 1997 to identify stable protein domains; and they will then stitch them back together in the correct order with an enzyme found in pathogens (such as Staph aureus), which attaches proteins to the surface of a cell.
Boder’s technique includes a method called “yeast surface display.”
First, they randomly fragment the CesA protein into hundreds of pieces; then, use genetic engineering to fuse the CesA fragments with a yeast protein destined for the surface of the cell, where it can be detected by a tag that fluoresces under a laser beam.
“We put these into the yeast,” Boder says. If the yeast allows the CesA fragment to accompany the protein through its secretory pathway to the surface of the cell that means it is folded, stable, and soluble—an unadulterated domain—part of the working whole. If not, the yeast detaches the fragment from the fused protein and recycles it.
While this part of the project should prove useful to O’Neill’s work, for Boder, it’s reapplying a tool they already have to a new problem. A second part of their project will use the S. aureus enzyme to reassemble domains to allow structural studies by neutron scattering.
“In the longer term what really interests me is engineering more than one enzyme and tag, so that each stable domain has it’s own enzyme and tag marker,” Boder says. “Individual tags and enzymes that match up with neighboring tags and enzymes will allow us to bring the pieces back together in the proper order.”
Domain identification and enzymatic ligation for structural biology of complex proteins
Eric Boder, UT Chemical and Biomolecular Engineering Department
Structural biology of metabolic and signaling pathways in plants
Hugh O’Neill, ORNL Biology and Soft Matter Division