Objective
DEVELOPMENT OF BASIC RESEARCH METHODS IN A MODEL STUDY OF HOW ALTERATIONS IN THE PROTEIN STRUCTURE CAN CHANGE FUNCTIONS.
THE ELONGATION FACTOR -TU IS AN INTERESTING MODEL BECAUSE OF ITS MULTIFONCTIONS IN PROTEIN BIOSYNTHESIS AND VIRAL RNA REPLICATION. ITS X-RAY STRUCTURE IS ALREADY ANALYZED AND IT COULD BE AN INTERESTING MODEL FOR MANY OTHER BIOLOGICALLY IMPORTANT GTP-BINDING PROTEINS (FOR EXAMPLE THE ONCOGENIC RAS GENE PRODUCT OR THE TRANSDUCING PROTEIN).
IN LONG TERM, THIS STUDY COULD ALSO LEAD TO MORE ECONOMIC PRODUCTION OF THE ANTIBIOTIC KIRROMYCIN WHICH HAS ITS SITE OF ACTION ON THE ELONGATION FACTOR-TU AND IN THE DESIGN OF NEW DRUGS THAT SELECTIVELY BINDS TO THE ELONGATION FACTOR -1 WHICH IS THE EUCARYOTIC ANALOG OF THE BACTERIAL FACTOR -TU.
The developments of the last decade in the determination of protein structure and the mutation and production of proteins have begun to make feasible the design of new proteins not from scatch, but by modifying their structure in small steps. However, in order to exploit this possibility, much research into the basic laws that govern the structure and function of proteins is still needed.
Research was carried out in order to see how alterations in a protein's structure can change its function, its stability or other properties. This needed expertise in protein structure molecular genetics sitedirected mutagenesis and in functional testing. The chosen 'model' protein was the elongation factor EF-Tu of Escherichia coli. This multifunctional enzyme, then known to play a central part in protein biosynthesis, has since gained in importance as the only really thoroughly studied representative of the rapidly expanding class of known guanosine triphosphate (GTP) binding proteins.
Knowledge of the protein's structure was used to predict the effect of single amino acid substitutions and the modified protein was made to see what could be learned from comparing the result with the prediction.
Results have provided confirmation of some early ideas about the connection between the spatial structure and the biochemical mechanism of the model protein EF-Tu. The first complete chain tracing was made of the nicked guanosine diphosphate (EF-Tu GDP) complex, in this case a difficult task. The unbroken protein was crystallized and the first X-ray data was collected. Mutagenesis work led to an understanding of the detailed tasks of the residues valine-20, proline-82 and histidine-84. It has been possible to prepare and study the GTP binding domain in isolation from the rest of the molecule, and also to crosslink EF-Tu chemically to its cellular partner transfer ribonucleic acid (tRNA). Genetic studies revealed a novel mechanism of regulation for this protein, with important implic ations for the synthesis of stable ribonucleic acid (RNA) and the control of bacterial growth.
SITE DIRECTED MUTAGENESIS OF THE ELONGATION FACTOR EF-TU.
IN PARTICULAR :
1. THE TUF GENE, CLONED IN PEMBL, WILL BE MUTAGENIZED BY USING SYNTHETIC OLIGODEOYNUCLEOTIDES AS MUTAGENIC PRIMERS IN ORDER TO MODIGY CYS-81 AND ASN-135.
2. THE PLASMID BORNE EF-TU WILL BE AMPLIFIED BY RUNAWAY EXPRESSION PLASMIDS OR SECRETION PLASMIDS.
3. THE MUTATED PROTEINS WILL BE CHARACTERIZED BY THE DIFFERENT EF-TU FONCTIONS (INTERACTIONS WITH GTP/GDP AND WITH AA-TRNA, KIRROMYCIN RESISTANCE).
4. OVER EXPRESSION AND CRYSTALLIZATION OF THE MUTATED PROTEINS.
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. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences molecular biology molecular genetics
- natural sciences biological sciences genetics mutation
- natural sciences biological sciences genetics RNA
- natural sciences chemical sciences organic chemistry amines
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
PALAISEAU
France
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