Objective
The goal of the project is to develop an innovative procedure for obtaining high-resolution structures of biomolecular complexes by NMR in solution. The approach is based on exploiting an existing binding site for paramagnetic ions in one of the biomolecules interacting in the complex or creating a suitable binding site by protein engineering. The presence of the paramagnetic metal induces NMR effects that only nowadays can be properly analyzed and provide the long range constraints that are needed to obtain the spatial relationships among the biomolecular components. In diamagnetic complexes the conventional constraints are relatively few and short range. This procedure will constitute a major advance with respect to the current state of the art. In the context of pharmaceutical industries, there is interest in high quality structural information on biomolecular complexes. For instance, structural characterization of protein-DNA complexes is desirable, in view of the need for constructing efficient carriers to take DNA into cells for gene therapy and DNA vaccination.
The procedure will be developed and tested on protein-DNA complexes, and lanthanide ions will be used as metal probes. Two well-characterized DNA binding dornains, representative of two different classes of DNA-binding proteins will be used. The first is a zinc-finger, in which we plan to convert the zinc binding site into a lanthanide binding site. The second is a protein lacking an intrinsic metal binding site, in which we plan to engineer a lanthanide binding site modeled after known calcium binding sites (e.g. EF hand motifs). Rational design strategies will be used. Once the lanthanide binding sites have been created, the proteins will be bound to their target DNA fragments. New NMR methodologies, only in part and very recently developed, will lead to the solution structure. The choice of working on protein-DNA is due to industrial interest. The industrial partner in this project plans to hybridize DNAbinding proteins with cell-penetrating proteins to achieve higher efficiency in delivering plasmids into the nuclei of eukariotic cells. The plasmids will carry the DNA fragment recognized by the DNA-binding protein. The presence of the paramagnetic probe will help in assessing the structural integrity of the whole hybrid-plasmid adduct. Once available, the lanthanide probe may be used at the industrial level to perform whole-cell imaging through appropriate spectroscopies (e.g. MRI, fluorescence), to allow the intracellular location of the hybrid-plasmid adduct to be determined. This latter possibility may be important in assessing reasons for low Mtes of combination of the inserted DNA and the host cell DNA.
Together with nuclear longitudinal relaxation rates and pseudocontact shifts, transverse relaxation rates and the newly observed orientation effects at high magnetic field (e.g. 800 MHz) will be exploited for the first time. All four effects will be, again for the first time, developed as a general methodology to solve the solution structure of biomolecular complexes. The success of the project will be easily assessed by the achievement of the following objectives: i) expression and purification of one or more DNA-binding proteins carrying an engineered lanthanide-binding site; ii) development and optimization of the NMR methodology to maximize the number of new constraints to solve the structure of biomolecular complexes by the use of different paramagnetic metal ions; iii) obtaining high quality NMR solution structure(s) of protein-DNA adduct(s). Objectives ii) and iii) are the main goals of this research, the first of general impact and the second specific. Objective ii) has the highest innovation content, and its achievement will hopefully constitute a breakthrough in the field.
The applicants believe that the project fully complies with the three technological requirements of Area 6 (Structural Biology): a) experimental determination of three dimensional structures, b) improvement of structure determination techniques and c) development of biochemical entities with the desired functions. In the present proposal, the prominent topic is topic b). The applicants also believe that the proposal fully meets the criteria of tMnsnational collaboration (Italy, England, The Netherlands), interdisciplinarity (spectroscopy, biochemistry, biotechnology), scientific and technical excellence and novelty (involvement of scientists from excellence centers in NMR, innovative NMR strategies), precompetitive character (full involvement of industry at the research and not at the development level), and wide possibilities of exploitation of results.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
- natural sciencesbiological sciencesgeneticsDNA
- natural scienceschemical sciencesinorganic chemistrytransition metals
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- natural sciencesbiological sciencesmolecular biologystructural biology
- natural sciencesphysical sciencesopticsspectroscopy
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Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
50121 FIRENZE
Italy