X-ray Crystallography Reveals New Insights into Pentapeptide Repeat Protein Structure
Results may help us harness cyanobacteria to sequester carbon
Results: A team of scientists from Pacific Northwest National Laboratory, Washington University in St. Louis and Brookhaven National Laboratory is investigating cyanobacteria because it could play a role in carbon sequestration, a method for removing carbon dioxide from the atmosphere. In the process they have determined the molecular structure of a second pentapeptide repeat protein (a.k.a. Rfr protein) from the cyanobacterium Cyanothece 51142.
To the researchers' surprise, the investigation revealed four structural features—ways protein are folded up within the cell—the scientific community had not previously observed for pentapeptide repeat proteins. These features, some of which are summarized in the figure, are 1) an Rfr-fold composed of only type II β-turns, 2) a 24-residue insertion, 3) a disulfide bracket and 4) a single residue bulge.
Pentapeptide repeat proteins are found primarily in bacteria, especially cyanobacteria. The combination of sheer numbers and diverse cellular locations within cyanobacteria points to these proteins having important, as yet unknown, biochemical and physiological functions in cyanobacteria. The newly uncovered structural features may help the scientific community uncover the biological role of pentapeptide repeat proteins within the cell.
Summary of the steps to determine the crystal structure for Rfr23. The first step is to grow a crystal, shown here highly magnified for Rfr23. X-ray diffraction data is then collected on this crystal at a synchrotron and the diffraction data converted into an electron density map. The structure of Rfrr23 is then “built” into the electron density map. Here, the structure is presented as a cartoon colored spectrally (red to violet) from the C-terminus. The structure is dominated by an Rfr-fold, composed of nearly six complete coils, C1-C6, that is capped at the N-terminus with a small a-helix. Enlarged View
Proteins that play significant roles in cyanobacteria functions are being identified by genomic, proteomic, transcriptomic and metabolomic data collected at PNNL and at Washington University as part of an EMSL Membrane Biology Scientific Grand Challenge.
Why it matters: Cyanothece is an intriguing organism that uses circadian rhythms to perform photosynthesis during the day and nitrogen fixation at night. By absorbing carbon dioxide during the day and converting it to organic carbon and atmospheric oxygen, the cyanobacteria sequester carbon naturally. Answering fundamental questions about the bacterium's biology could provide the information needed to enhance its carbon sequestration abilities.
Understanding the structure, function and dynamics of pentapeptide repeat proteins may help us understand how cyanobacteria regulate energy transduction, photosynthesis, hydrogen production, nitrogen fixation, and metal ion homeostasis. Ultimately, the goal is to use this information to engineer oxygenic photosynthetic microbes with enhanced carbon sequestration abilities.
Methods: The crystal structure of Rfr23 was determined at 2.1 Å resolution using selenomethionine-labeled crystals grown at PNNL and data collected at the National Synchrotron Light Source at BNL. The coordinates for this structure have been deposited in the RCSB Protein Data Bank. While the crystal structure analyses revealed interesting new details on the three-dimensional structure of a pentapeptide repeat protein, such analyses were complemented by biophysical studies of the protein in solution using the suite of nuclear magnetic resonance spectrometers at the Environmental Molecular Sciences Laboratory at PNNL. One finding was that the disulfide bracket is crucial to the integrity of the entire structure. The disulfide bracket is formed between C39 and C42 and bridges the β-turn between the first and second pentapeptide repeat in the first coil of the Rfr-fold. "Fingerprint" spectra for 15N-labeled Rfr23, collected in the absence and presence of dithiotreitol, a chemical that destroys disulfide bonds, showed that the entire protein became unstructured after the chemical bond's destruction.
What's next: The ultimate goal is to determine the biological function of pentapeptide repeat proteins. Biochemical studies in pursuit of this goal are under way. These will be aided by solving the structures of additional PRPs whose structures may reveal yet additional insights into the structural variation within these fascinating group of proteins.
Acknowledgments: The research team includes Dr. Garry Buchko, PNNL; Dr. Howard Robinson, Brookhaven National Laboratory; Dr. Himadri Pakrasi, Washington University; and former PNNL staff member Dr. Mike Kennedy. This work is part of an EMSL Membrane Biology Scientific Grand Challenge project at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research program located at PNNL.
Buchko GW, H Robinson, HB Pakrasi, and MA Kennedy. 2008. "Insights into the structural variation between pentapeptide repeat proteins—Crystal structure of Rfr23 from Cyanothece 51142." Journal of Structural Biology (In Press - Available online).
Buchko GW, S Ni, H. Robinson, EA Welsh, HB Pakrasi and MA Kennedy. 2006. "Characterization of two potentially universal turn motifs that shape the repeated five-residues fold—crystal structure of a luminal pentapeptide repeat protein from Cyanothece 51142." Protein Science 15:2579-2595.
Buchko GW, S Ni, H. Robinson, EA Welsh, HB Pakrasi and MA Kennedy. 2006 "Cloning, expression, crystallization and preliminary crystallographic analysis of a pentapeptide-repeat protein (Rfr23) from the bacterium Cyanothece 51142." Acta Crystallographica F62(12):1251-1254.