Properties of Delta-Endotoxines
Studies of Molecular Basis of Specificity of Bacillus Thuringiensis delta-Endotoxins by X-ray Structural analysis and Model Analysis
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- AGR-PPR/Plant Protection/Agriculture
3 Approved without Funding
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Russian Academy of Sciences / Institute of Crystallography, Russia, Moscow
- University of Georgia, USA, GA, Athens
Project summaryThe main objective of the project is to study the three-dimensinal structures of a number of crystal proteins (CryIA(c)), CryIII, and CryV), or d-endotoxins of the bacterium Bacillus thuringiensis, that is used for biological control of insects and other crop pests, and of complexes of these proteins with receptors of target cells by X-ray structural analysis and model analysis and, based on the results obtained, to work out recommendations for protein engineering of d-endotoxins.
The project implies solution of the following problems:
- Development of methods for isolation and purification of receptors specific to CryIII and CryV d-endotoxins.
- Preparation of highly purified samples of d-endotoxins and their receptors.
- Studies of conditions for the formation (in solutions) of the complex between CryIA(c) and its receptor and those between CryIII and CryV and their receptors.
- Search for conditions of crystallization of d-endotoxins, receptors, and their complexes and preparation of crystals suitable for X-ray structural analysis (dimensions 0.2-0.4 mm) and of good quality (resolution higher than 2.5 A).
- Preliminary X-ray structural analysis of the crystals to estimate whether they are suitable for subsequent X-ray diffraction studies (symmetry, unit cell parameters, and maximum resolution).
- Collection of X-ray intensity data sets for the crystals suitable for X-ray structural analysis with the use of synchrotron radiation at the highest possible resolution.
- Calculations by the molecular replacement method to establish the three-dimensional structure of the crystals. Wherever necessary, preparation of heavy-atom derivatives and calculations by the multiple isomorphous replacement method.
- Analysis of the three-dimensional structure of d-endotoxins, receptors, and toxin-receptor complexes determined in our studies and reported in the literature to reveal the regions of the molecules determining their specific binding.
- Comparative analysis of the known amino-acid sequences of d-endotoxins and receptors in the light of the three-dimensional structure studied. Identification of the amino-acid sequences of the determining regions of d-endotoxin molecules, which may be responsible for their specific binding to various receptors, by using model analysis.
Successful determination of the structure of proteins under study and their complexes will make it possible to reveal the molecular basis of specificity of toxin-receptor binding and, based on these data, to perform gene-engineering studies aimed at designing new producers of pesticides, obtaining transgene plants, and solving the problem of resistance of insects to d-endotoxins.
These studies will provide the basis of solving the applied problems and will also contribute substantially to the solution of the fundamental problem of protein-protein recognition.
This work is in line with the goals of the International Scientific and Technical Centre, as it enables skilled specialists of the State Research Center for Applied Microbiology, engaged earlier in studies of toxins of highly dangerous bacteria according to the program of bacteriological weapon defence, to direct their activity towards solution of fundamental and applied problems of agricultural plant and forest protection and promotes their integration into the international scientific community.
Potential Role of Foreign Collaborators
Foreign participants of the project are the University of Georgia (Athens, Georgia), the University of York (England), and the European Molecular Biology Laboratory (Hamburg, Germany).
The Laboratory of Toxins, University of Georgia (Head is Dr. M.J. Adang, Department of Entomology, University of Georgia, USA) is one of the world laboratories in studies of the structure and functions of B. thuringiensis toxins leading. It is expected that researchers of this laboratory will help in the project, first, by advice in this field and by providing access to the required scientific and technical information, second, by supplying a number of bacterial strains, cloned genes, and insects, and, third, by organizing and performing joint studies on the project, dealing with purification of proteins.
Institute of Crystallography, Russian Academy of Sciences has a long-standing experience in collaboration with research groups from the University of York and European Molecular Biology Laboratory (Head is Dr. K.S. Wilson), working in the field of X-ray structural analysis of proteins. It is expected that during performing joint studies, these institutions, as participants of the project, will provide unique equipment they possess for measurements and calculations.
The project participants invite other research, industrial and commercial organizations from USA, countries of European Union, Japan, Norway and Southern Korea to collaboration on the project.
The following forms of collaboration are possible:
- Participation in the development of work plan
- Exchange of information during project implementation
- Mutual review of technical reports
- Joint seminars and workshops
- Verification of results using independent methods and/or equipment
- Sharing of scarce materials, samples, resources
- Joint or parallel investigations
- Consultation on Intellectual Property Rights, in case of joint invention
It should be noted that, beyond collaboration in the forms mentioned above, governmental, intergovernmental, private, and commercial organizations might directly fund the project within the framework of the ISTC Partner Program. Information on the benefits of the Partner Program is available from the ISTC Secretariat.