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Marzluf Adviser Dr. Venkat Gopalan Department of Biochemistry UMI Number: 3011021 UMI UMI Microform 3011021 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17. United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Cry2Aa, a Bacillus thuringiensis 5-endotoxin, is specific to both dipteran and lepidopteran insects. Upon digestion by gypsy moth {Lymantria dispar) midgut proteases, the first 49 amino acids at the N-terminus are removed from the 63 kDa protoxin, to form a 58-kDa active fragment. The protease-resistant core is comprised of ValSO as the first amino acid, which is located at the loop between the aO and a1 helices of domain I. The toxin’s potency was lost when further in vitro midgut protease processing continued. The loss in toxicity was caused by proteolytic cleavage at the carboxyl end of Leu 144, which is on the loop between aS and a4 of domain I. To prevent the production of the non-toxic fragment, five mutant proteins, L144D, L144A, L144G, L144H, and LI44V, were constructed. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. However, the mutant proteins were as toxic to the insects as the wild type protein, indicating that the cleaved fragment is associated with the rest of the entire protein molecule in the midgut environment. The cleavage was also protected when the toxin is bound to the midgut membrane. The role of the 49-amino acid length of the N-termina! sequence of Cry2Aa was investigated. This short peptide sequence contains one extra a helix, called aO, which is located between Phe24 and His46. Gene deletion and site-directed mutagenesis results indicated that the presence of this a helix is necessary for the formation of biological active inclusion of Cry2Aa. Deletion of the cry2Aa gene at the aO helix, and deletion of the entire N-terminus region led to production of the non-soluble inclusion bodies that were non-toxic to both L. dispar and A. quadrimacuiatus. However, expression cry2Aa gene that contains the aO helix, but lacking the Val3 - Asn17 region, gave the soluble, and toxic crystal inclusions. Tertiary structure of Cry2Aa reveals a number of intramolecular and intermolecular interactions contributed by the amino residues in aO. The intramolecular interactions might stabilize, and facilitate folding of the Cry2Aa protoxin, as demonstrated by the observation that the melting temperature of the Cry2Aa protoxin and the mutant protein containing aO were 7 °C higher than the active Cry2Aa fragment. Mutation of amino acids involved in intramolecular interactions lowered the yield of the inclusion bodies. Loss in the inclusion amount was also found when the residues involved in intermolecular bondings were mutated. Finally, results from intensive site-directed mutagenesis of amino acids on loop 1 and loop 2 of domain II and bioassays against A. quadrimacuiatus and L dispar revealed that specificity of Cry2Aa to dipteran insects was conferred by amino acids in loop 1, while the amino residues on loop 2 are responsible for Lepidoptera specificity. The mutant proteins A2 (F 320-321AAA), F320A, and P321A were less toxic to A. quadrimacuiatus larvae. Change in toxicity was related to change in competition binding affinity. However, the same mutant proteins were as toxic to L dispar larvae as the wild type protein and change in binding properties to L. d/spar BBMV was not observed. In contrast, the mutant proteins obtained from the alterations in the region, i. e., A5 (DRE 383-5AAA), D383V, R384A, and E385A, were less toxic to only L dispar. Results from dissociation experiments indicated that these mutant proteins were not involved in irreversible binding. The dramatic changes found in these mutant proteins were reversible binding. Therefore, Initial binding of amino acids on loop 1 and loop 2 of Cry2Aa might be a factor that determines the toxin dual specificity. The obtained results concerning active fragment processing, solubility, and specificity of Cry2Aa might reveal a fundamental mode of action of Cry2Aa and other Cry toxins from Bacillus thuringiensis. Active fragments of Cry toxins seem to contain all seven a-helices to retain their biological activity. The results also indicated that formation of the bioactive crystal in the bacterial cells requires not only the active fragment portion, but also the extra-portion of the protoxins. These peptide portions might facilitate both protein folding and assembly of the protoxin molecules. Finally, the results obtained from the Cry2Aa-receptor binding and toxin specificity experiments might help in engineering more potent and more specific Cry toxins, which will be used to control the certain target insects. iv Dedicated to my father ACKNOWLEDGMENTS I wish to express my sincere appreciation to my advisor, Dr. Donald H. Dean for his guidance and constant inspiration throughout my graduate study. I am especially grateful to him for his patience and understanding the situation I have had during staying in this school. I gratefully acknowledge Dr. George Marla, and Dr. Venkat Gopalan for their helpful suggestion and constructive criticism on my dissertation, and their serving in my committee. I thank Dr. Tipvadee Attathom, and Dr. Supat Attathom, who gave me a chance to continue my studies in the US. I would like to thank the Royal Thai Government for financial support during the early period of my study. I thank Dr. Daniel Ziegler, from Bacillus Stock Center, for providing some financial support and information about Bacillus thuringiensis. I gratefully acknowledge Dr. Richard Morse and Dr. Robert Stroud, University of California, San Francisco, for kindly providing the very helpful information of Cry2Aa structure. I thank Dr. Algimantas Valaitis, USDA, Delaware, Ohio, for protein sequencing. VI I thank Frank Martin and Win McKlane, from USDA, Massachusetts, for kindly providing gypsy moths. I thank April Curtiss to her assisting in almost everything during my staying in this lab. I also thank Joy, Chung-Wen Wu for her assistance in protein purification and gypsy moth bioassays. I thank Mohd Amir Abdullah for mosquito samples, and Norapan Kittivat for Cry toxin alignment diagrams. I thank Dr. Oscar Alzate for biophysics experimental data and Dr. Mark Foster for the protein unfolding studies facilities. I am grateful to Dr. Mi K. Lee, Dr. Taek You for some suggestion and making a friendly environment in this lab. Many thanks to my former and present laboratory colleagues for their help and best wishes: Dr. Jeremy Jenkins, Sang Soo Oh, Sylvia Liu, Marry Beth Dunn, and Carroll Ziegler. Special thanks go to Tara Grove for her friendly help in everything. Finally, I thank my family members who took care of my father during 5 years of my staying in the US. VII VITA May 13, 1966 ......................................................Born-Mukdahan, Thailand 1984 - 1988 ........................................................8 . S. Biology Khon Kaen University Khon Kaen, Thailand 1988 - 1992 ........................................................M.S., Biochemistry Mahidol University Bangkok, Thailand 1992 -1 9 9 5 ........................................................Research associate National Center for Genetic Engineering and Biotechnology Bangkok, Thailand 1995 - 1997 ........................................................ Fellowships Royal Thai Government 1997 - 2001 .......................................................

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