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Biological Sciences Division
Research Highlights

July 2009

On the Light Path to Bioenergy

High-sensitivity proteomics lead to discovery of operon in O2-producing protein

cyanobacteria
Representation of the slr0144-slr0152 operon in (A) Synechocystis 6803 and (B) other cyanobacteria. In A, the black arrows indicate open reading frames whose products were observed to be differentially expressed in PSII complexes isolated from various mutant strains. In B, the slash indicates genes are not clustered. X indicates that there is no ortholog, or similar gene, in the genome. The numbers correspond to the gene designation or contig number in various cyanobacteria. Enlarge Image.

Results: Researchers from Pacific Northwest National Laboratory and Washington University in St. Louis discovered a novel cluster of genes that encode proteins essential for green plants to thrive. They identified six proteins linked to a complex called photosystem II (PSII), which forms a cluster of nine genes—slr0144 to slr0152. The researchers named the proteins Pap, for PSII assembly proteins.  They identified these components as well as proteins associated with PSII throughout its life cycle, in functions such as assembly, repair, or degradation.

Photosynthesis is the process by which all green plants and some algae use sunlight to synthesize organic compounds from carbon dioxide and water.  One of the initial stages in the chain of photosynthesis involves PSII, the membrane protein complex found in organisms such as green plants, green algae, and cyanobacteria, otherwise known as blue-green algae.

PSII is a light harvesting system that splits water molecules to produce oxygen.  An operon is a cluster of structural genes found in bacteria that are expressed and regulated as a unit. It consists of a promoter and an operator; a promoter is the region of the operon that acts as an initial binding site during gene transcription. The operator is a segment of DNA that regulates the activity of the structural genes of the operon, somewhat like an on/off switch.

"We identified additional key components to PSII involved in assembly and function," said Dr. Jon Jacobs, a protein chemist at PNNL. "The basic mechanisms are known, but there are a lot of details that we don't know about. This research can lead to a better understanding of how these proteins work in other electron transport chain mechanisms, which could alter how we view this entire process."

Why it matters: Future clean energy will rely heavily on efficient conversions of light energy into biofuel.  Dramatic progress has been made in recent years in understanding the fundamental reaction of photosynthesis.  Scientists continue to study photosynthetic organisms such as cyanobacteria for their potential as bioenergy sources.  However, to get to that point, much more must be known about the processes surrounding photosynthesis.  Previously, it was difficult to identify proteins associated with PSII because of their relatively short association periods.  A high-sensitivity proteomics approach allows a more comprehensive investigation of proteins involved in the complex assembly of PSII.

Methods: The researchers used high-throughput proteomics at the U.S. Department of Energy's Environmental Molecular Sciences Laboratory, a national scientific user facility at PNNL, to study the composition of isolated PSII complexes. They compared protein profiles from mutants of a strain of cyanobacterium Synechocystis 6803. To deduce the role of the Pap operon proteins on cellular function, the researchers performed a "knockout" of the entire operon.  A knockout is a technique for deleting or inactivating a gene to determine what role the gene plays by its absence.   The researchers observed a measurable decrease in PSII water oxidation caused by a loss of functional PSII complexes, implying a role in stabilizing assembly/degradation events through key binding interactions.

"In this case, not much happened to the core functions after deletion, but a measureable loss in overall water oxidation was observed because of a reduction in the total number of functional PSII complexes," Jacobs said.  "This implies that the PSII complexes are not optimized in the assembly, and/or when they are damaged they aren't getting repaired effectively.

What's next: Studies revealing that the cofactor binding sites of Paps are functional and that they are able to transfer cofactors—non-protein compounds required by enzymes to function—could provide insight into how they are assembled into the complex. Research also will focus on whether Paps are significant to PSII assembly or if they help in assembly of other complexes in the electron transport process.

"This is important research at the discovery level," Jacobs said.  "Greater understanding of these systems will be critical for any downstream work in altering or modifying the organism and or complex, among other things."

Acknowledgments: The research team is Jon Jacobs, Kim Hixson, Matthew Monroe, David Camp II, and Richard Smith, PNNL; Kimberly Wegener, Eric Welsh, Leeann Thonton, Nir Keren, and Himadri Pakrasi, Washington University, St. Louis. This work was supported by EMSL and by the National Science Foundation.

Reference: Wegener KM, EA Welsh, LE Thornton, NS Keren, JM Jacobs, KK Hixson, ME Monroe, DG Camp II, RD Smith, and HB Pakrasi. 2008. "High Sensitivity Proteomics Assisted Discovery of a Novel Operon Involved in the Assembly of Photosystem II, a Membrane Protein Complex." Journal of Biological Chemistry 283(41):27829-27837.


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