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Protein engineering and biophysical analysis of insecticidal toxins is the major research interest in my laboratory. The ultimate goal of these studies is to understand the structure/function relationships of these proteins to create more specific and environmentally safer pesticides.
One project involves locating the functional domains of insecticidal crystal proteins (ICPs) of Bacillus thuringiensis. Site-directed mutagenesis and homology switches of portions of genes which display different insecticidal activity have created a set of mutants which allows us to identify the "specificity domains" on two ICPs. Intensive site-directed mutagenesis will reveal which amino acids are involved in the insecticidal activity.
Biophysical analysis of these proteins probes the mechanism of action of the ICPs. The interaction of the toxins with membrane receptors is being analyzed by kinetic analysis of binding and location of the binding sites using brush border membrance vesicles and insect tissue culture cell. Purificaton and analysis of receptors are one aspect of this project; mutations of the toxin protein to analyze the protein-protein interaction of toxin-receptor binding is another. Electrophysiological techniques (patch and voltage clamps) are used to measure the effects of mutations on the ion channel function of ICP. The conformational changes that take place upon receptor binding and partitioning into the membrane is being studied by site-directed spin labeling using electron paramagnetic resonance (EPR).
We also examine expression of insecticidal proteins in foreign organisms in my laboratory. Overexpression in E. coli allows mutant proteins to be produced to greater than 50 percent of total cellular protein allowing rapid purification of protein for biochemical study and insect bioassay. In addition, we are interested in the expression of ICPs in plants and algae, in collaboration with other scientists at Ohio State and elsewhere.
Alzate O, You T, Claybon M, Osorio C, Curtiss A, Dean, D.H.. (2006) "Effects of disulfide bridges in domain I of Bacillus thuringiensis Cry1Aa delta-endotoxin on ion-channel formation in biological membranes."Biochemistry. 45(45), 13597-605.
Dean, D.H.. (2006) "Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus". BMC Biochem. 7, 16.
Liu XS, Dean, D.H.. (2006) "Redesigning Bacillus thuringiensis Cry1Aa toxin into a mosquito toxin." Protein Eng Des Sel. 19(3), 107-11.
Padilla C, Pardo-Lopez L, de la Riva G, Gomez I, Sanchez J, Hernandez G, Nunez ME, Carey MP, Dean, D.H., Alzate O, Soberon M, Bravo A. (2006) "Role of tryptophan residues in toxicity of ry1Ab toxin from Bacillus thuringiensis." Appl Environ Microbiol. 72(1), 901-7.
Liu XS,Dean, D.H. (2006) "Redesigning Bacillus thuringiensis Cry1Aa toxin into a mosquito toxin.", Protein Eng Des Sel. 19(3), 107-11.
Abdullah MA,Dean, D.H. (2004) "Enhancement of Cry19Aa mosquitocidal activity against Aedes aegypti by mutations in the putative loop regions of domain II.", Appl Environ Microbiol. 70(6), 3769-71.
Abdullah MA, Alzate O, Mohammad M, McNall RJ, Adang MJ, Dean, D.H. (2003) "Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering.", Appl Environ Microbiol. 69(9), 5343-53.
Dean, D.H., T.H. You, F. Rajamohan, M.K. Lee, J.L. Jenkins, M. Audtho, S.-J. Wu and X. Liu. (2002). "Rational design of Cry toxins. (In. R.J. Akhurst, C.E. Beard and P.A. Hughes, eds) Biotechnology of Bacillus thuringiensis and Its Environmental Impact." Scribbly Gum Publications. Canberra, Australia.
Daniel, A., Sreedhara Sangadala, S., Dean, D.H. and Adang, M.J.( 2002) "Denaturation of either Manduca sexta aminopeptidase N or Bacillus thuringiensis Cry1A toxins exposes binding epitopes hidden under non-denaturing conditions." Appl. Environ. Microbiol. 68,2106-2112.
Alcantara, E., O. Alzate, and Dean, D.H. (2001). "Roll of a-helix seven of Bacillus thuringiensis Cry1Ab delta-endotoxin in membrane insertion, structural stability and ion channel activity and toxicity." Biochemistry 40: 2540-2547.
Arnold, S., A. Curtiss, Dean, D.H., and O. Alzate. (2001). "The role of a proline induced broken-helix motif in a-helix 2 of Bacillus thuringiensis delta-endotoxins." FEBS Letts. 490: 70-74.
Jenkins, J.L. and Dean, D.H. (2001). "The complex receptor-binding interactions of insecticidal proteins from Bacillus thuringiensis." BIAjournal 8: 25-27.
Valaitis, A. P. J.L. Jenkins, M.K. Lee, Dean, D.H. and K.J. Garner. (2001). "Isolation and Partial Characterization of Gypsy Moth BTR-270, an Anionic Brush Border Membrane Glycoconjugate that Binds Bacillus thuringiensis Cry1A Toxins with High Affinity." Archives of Insect Biochemistry and Physiology 46:186-200.
Lee, M.K., J.L. Jenkins, T.H. You, A. Curtiss, J.J. Son, M.J. Adang and Dean, D.H. (2001). "Mutations at the arginine residues in a8 loop of Cry1Ac Bacillus thuringiensis delta-endotoxin affect toxicity and binding to Manduca sexta and Lymantria dispar Letts aminopeptidase N." FEBS. 497: 108-112.
Daniel, A., Dean, D.H. and M.J. Adang. (2001). "Analyses of membrane permeabiliziing functions of Bacillus thuringiensis Cry1Ab domain I and domain II mutant toxins using light scattering technique." Pest Biochem Physiol. 70: 7-18.
Banks, D.J., J.-L. Jurat-Fuentes, Dean, D.H. and M.J. Adang. (2001). "Bacillus thuringiensis Cry1Ac, and Cry1Fa d-endotoxins binding to a novel 110-kDa aminopeptidase in Heliothis virescens is not GalNAc mediated." Insect Biochem Molec Biol. 31: 909-918.
Jenkins, J.L and Dean, D.H. (2001). "Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors." BMC Biochem. 2:12.