Progress 11/01/99 to 10/31/04
Outputs
During this grant period, we have expressed the pheromone binding protein in bacteria, refolded using 4 different methods (manuscript No. 3) and purified from inclusion bodies. The protein was also sub-cloned and expressed for periplasmic expression in pET 22b (+) vector. It has been reported that the disulfide bonds are formed correctly when such proteins are expressed in the periplasm. I have also expressed the protein in Origami cells. The Origami hosts possess a mutation in the glutathione reductase (gor) gene, which is one of the central enzymes in cytoplasmic disulfide metabolism. Origami strains therefore promote higher levels of disulfide bonding in the E.coli cytoplasm. We have been able to express this PBP protein as a soluble protein in the Origami strain. All these above protocols were tried to get the highest yield of native protein since we needed 10-15 milligrams of pure native protein in labeled form. 15NH4Cl and particularly (13C6) glucose are very
expensive. We have purified all these proteins first on the basis of charge (anion exchange) and then on the basis of size (gel filtration) on a FPLC system. The activity of the protein was tested by photoaffinity labeling using a tritium labeled pheromone analog. I have collected all 3 and 4-dimensional NMR data sets (HNCA, HNCO, HNCACB, HNCACO, HSQC, HSQC-NOESY, CBCACONH, CC(CO)NH and HCACO etc). I have processed all these data sets using NMRPipe and NMRDraw programs. The protein has been completely sequence specifically assigned manually using all the above NMR experiments (manuscript No. 1). 3-dimensional structure of the protein has been determined (manuscript no. 3). The ApolPBP structure is the first structure of a pheromone binding protein that binds or recognizes acetate pheromone and provides new insight in pheromone recognition through a comparative analysis with the alcohol binding PBP from B.mori. I have also studied the effect of pH and temperature on this protein both
by CD and NMR. I have also collected many multidimensional NMR experiments on the ApolPBP at acidic pH to understand the conformational switch and have initiated the resonance assignments process for this conformation. I have initiated three other projects and have made significant progress. One of these projects deals with structural and functional characterization of yeast oligosaccharyltransferase (OT). We have been able to determine the first 3-dimensional structure of an eukaryotic OT subunit, Ost4p, in model membrane solvent system (manuscript No. 4). The 3rd project is on the characterization of glutaminase interacting protein (GIP) from human brain. GIP is a small (124 residues) protein. The GIP protein containing a PDZ domain, acts as a scaffold for the function of the enzyme glutaminase. We have sub-cloned GIP into pET3c vector, overexpressed in BL21 DE3 cell lines and purified to homogeneity. The activity was tested through in-vitro pull down assay. Wehave prepared labeled
protein and collected 3-dimensional heteronuclear experiments. We have also characterized this protein by CD and fluorescence spectroscopy
Impacts
Characterization of the 3-dimensional structure of the protein and its binding site to pheromone, will help us in designing anti-pheromone or pheromone mimetics. Such pheromone mimetics or inhibitors can block the signal transduction pathway in moth olfaction. This can have great success in our ability to interfere in moth mating and there by controlling these insect pests through an environmentally friendly and selective way for biorational crop protection.
Publications
- 1. Smita Mohanty, Sergey Zubkov and Ramon Campos-Olivas (2003). 1H, 13C and 15N backbone assignments of the pheromone binding protein from the silk moth Antheraea polyphemus (ApolPBP). J. Biomol. NMR, 27, 393
- 2. Smita Mohanty and Sergey Zubkov, NMR assignments and secondary structure of the silk moth Antheraea polyphemus pheromone binding protein ApolPBP, Protein Science, volume 12, 121(2003).
- 3. Smita Mohanty, Sergey Zubkov and Angela Gronenborn, The solution NMR structure of Antheraea polyphemus PBP provides new insight into pheromone recognition by pheromone binding proteins, Journal of Molecular Biology (vol. 337, issue 2, page 443-451, (2004).
- 4. Sergey Zubkov, William J. Lennarz and Smita Mohanty, Structural basis for the function of a novel minimembrane protein subunit of yeast oligosaccharyltransferase (Proc. Natl. Acad.Sci. USA 101, issue 11, page 3821-3826 (2004).
- 5. Kimberly Lai, Sofya Maslyanskaya, Natalia Gutierrez and Smita Mohanty, Characterization of the C-terminal domain of the BLM protein, Protein Science, 13, 147 (2004).
- 6. Zarina Nestor, Smita Mohanty, Sergey Zubkov and Sofya Maslyanskaya, Biophysical characterization of the human Glutaminase-Interacting protein containing a PDZ domain and its interactions with C-terminal sequence motif of L-glutaminase, Protein Science, 13, 148 (2004).
- 7. Smita Mohanty, Sergey Zubkov and Angela Gronenborn, Structural characterization of the Anthraea polyphemus pheromone binding protein in acidic pH, Protein Science, 13, 234 (2004).
|
Progress 12/01/99 to 11/30/00
Outputs
This is the first annual progress report. This grant is funded for the structural characterization of the pheromone binding protein (PBP) from the silk moth Anthraea polyphemus. The size of the protein is 16 kDa. During this 1 year period, the protein has been expressed in E.coli in minimal media producing N-15 labeled and N-15, C-13 double labeled protein in over 20 mg quantities for solution NMR studies. The whole set of multidimensional NMR experiments required for the structural characterization of the protein, have been collected. These are 2- or 3- or 4-dimensional HSQC, HNCA, HNCO, HNCACB, CBCACONH, HSQC-NOESY, HCACO, HMQC, CCCONH and HCACO. These data have been processed with NMRPipe program and work is in progress for the proton, nitrogen and carbon resonance assignment for the protein backbone.
Impacts
Characterization of the 3-dimensional structure of the protein and its binding site to pheromone, will help us in designing anti-pheromone or pheromone mimetics. Such pheromone mimetics or inhibitors can block the signal transduction pathway in moth olfaction. This can have great success in our ability to interfere in moth mating and there by controlling these insect pests through an environmentally friendly and selective way for biorational crop protection.
Publications
- No publications reported this period
|