Research Interests

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Bioremediation of Environmental Manganese Using the Green Algae Chlamydomonas- Manganese is a trace metal pollutant that can enter the environment through acid mine drainage, automobile fuel additives, and metallurgical processes (such as welding). High concentrations of manganese in humans are a serious health concern and have been in implicated in a number of diseases. Currently, there is much work being done on the removal of cadmium, lead, and chromium from the environment using biotechnology. We propose to engineer a high affinity manganese binding protein in the algae Chlamydomonas reinhardtii that can be used in the bioremediation of manganese pollution. This approach has the advantage of producing a simple, cost-effective and highly selective technique to remove manganese contamination.

In this pilot study, two research projects will be initiated: 1) to develop strains of Chlamydomonas that binds manganese and 2) to develop a promoter system in Chlamydomonas that will allow tightly controlled expression of foreign genes. A commercially available phage display library will be screened for phages that contain manganese-binding peptides. After identifying phages that bind manganese, the DNA that encodes the manganese binding peptide will be sequenced, and a gene containing single and multiple tandem repeats of this manganese binding domain will be created. This gene will be expressed in Chlamydomonas, and targeted to the outer membrane by fusing this construct to the N terminal domain of proteins expressed in this region. The manganese binding of these Chlamydomonas strains will be assessed, and small-scale pilot tests, directed at purifying dilute concentrations of manganese from aquatic samples, will begin. In addition to these studies, an inducible promoter for the expression of this gene and other foreign genes in Chlamydomonas, based on the nitrate reductase promoter, will be developed.

At the end of the 18-month period, we hope to isolate a manganese binding peptide, to have a manganese-binding gene expressed in Chlamydomonas, and to begin pilot studies on the feasibility of the transgenic algae to bind manganese from polluted water. We also hope to have a tightly controlled expression system in Chlamydomonas to turn off and on this gene, as well as other foreign genes, by manipulating nitrate and ammonia levels in the growth media. Using genetically modified Chlamydomonas as a bioremediation agent has great promise since no toxic chemicals are necessary for producing this algal bioadsorbent in this organism and the cost of growing up large quantities of Chlamydomonas is small. .

 

Photosystem II- The reaction mechanism of plant water oxidation has attracted and continues to attract intense scholarly activity. It is among the most oxidizing reactions that occur in nature, yet there is no consensus mechanism in the field for how this reaction occurs. The determination of the molecular structure for the bacterial reaction center, a reaction center that shares much structural similarity to photosystem II (PSII), has done much to stimulate interest in the field and resulted in the Nobel Prize for Drs. Michel and Deisenhofer. Photosystem II also has an important role in the environment because it produces the atmospheric oxygen necessary for respiration in organisms. Photosystem II is part of the photosynthetic electron transport chain that uses two reaction centers in the transfer of electrons from water to NADPH+; it is a multi-subunit protein complex that uses light energy to oxidize water and to form molecular oxygen. Hydrophobic subunits of PSII, such as the D1 and D2 proteins, bind most of the prosthetic groups involved in the initial electron transfer events. Water oxidation occurs at a catalytic site containing four manganese atoms. The catalytic site accumulates the four oxidizing equivalents required for water oxidation. The intermediate states of the water oxidation cycle are called the S states (S0-S4). A low-resolution structure of a Chlamydomonas PSII reaction center, capable of water oxidation, does exist and will be helpful in our project.

There are three protein subunits that are associated with the water oxidation site, and are believed to function in protection of the manganese and in binding the calcium and chloride cofactors necessary for high rates of oxygen production. The subunits are named after their apparent molecular mass and are called the 33 kDa protein, the 24 kDa protein, and the 18 kDa protein, encoded by the psbO, psbP, and psbQ genes respectively. Of these, the 33 kDa protein, also known as the manganese stabilizing protein (MSP), is the most intensively studied and plays an important role in water oxidation. All these proteins are nuclear-encoded, and there probably are two copies of the manganese stabilizing protein per PSII reaction center. All the extrinsic subunits can be removed from photosystem II by biochemical treatments, and rebound to photosystem II, restoring oxygen evolution. This important finding allows us to reconstitute proteins both with site-directed mutations and with site-directed spin labeled amino acids. The specific aims of our overall research project will be to investigate how the extrinsic proteins function in water oxidation, and these aims are divided into three parts: 1) Identification of amino acid residues that are important for binding the extrinsic proteins to photosystem II; 2) Discovery of the amino acid residues of the manganese stabilizing protein that are involved in cofactor binding or that are important for efficient S state transitions; 3) Use site-directed spin labeling and electron paramagnetic resonance spectroscopy to probe structure/function relationships among the subunits of photosystem II. In this ND EPSCoR Seed Grant proposal, the focus will be on aim 3, site-directed spin labeling of the extrinsic proteins, and the development of a genetic screen to study the manganese stabilizing protein, aim 2.

From our previous research on the manganese stabilizing protein, using difference Fourier transform infrared spectroscopy (FT-IR); a catalytic role for the MSP in the water oxidation cycle was identified. Our ability to make structural changes in the manganese stabilizing protein from Chlamydomonas should lead to the identification of residues that are necessary for efficient catalytic turnover of photosystem II. The proposed research will identify the structurally and functionally important amino acids of the extrinsic proteins and give us a clearer picture of the role of these proteins in the water oxidation reaction. This research should also lead to a greater understanding of the protein-protein interactions of membrane proteins, an abundant class of proteins that are encoded by up to 40 percent of the genes in some genomes.

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