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CONSTRUCTION AND BIOLOGICAL FUNCTION OF ALTERED PROTEINS DEFINED BY THEIR SPATIAL STRUCTURE

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. IT X-RAY STRUCTURE IS ALREADY ANALYSED 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 A LONG TERM, THIS STUDY COULD ALSO LEAD TO MORE ECONOMIC PRODUCTION OF THE ANTIBIOTIC KIRROMICYN WHICH HAS ITS SITE OF ACTION ON 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.
1.- STUDY OF THE REGULATION OF THE EXPRESSION OF ELONGATION FACTORS -TU AND LALPHA ENCODING GENES.
2.- SITE AND SEGMENT DIRECTED MUTAGENESIS OF THE E.COLI TUFA AND TUFB GENES WHICH ENCODES THE ELONGATION FACTOR-TU..
3.- ISOLATION OF MONOCLONAL ANTIBODIES RAISED AGAINST TRYPTIC PEPTIDES OF THE ELONGATION FACTORS -TU AND LALPHA.
4.- CLONING AND MANIPULATION OF THE GENES CODING FOR THE ELONGATION FACTOR LA FROM TRYPANSOSOMES.

THE INFORMATION GATHERED FROM THESE APPROACHES WILL BE USED TO STUDY THE FUNCTIONS/STRUCTURE RELATIONS OF THE ELONGATION FACTORS.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

RIJKSUNIVERSITEIT LEIDEN
Address
Department Of Biochemistry Wassenaarseweg 64
2333 AL Leiden
Netherlands

Participants (2)

AARHUS UNIVERSITY
Denmark
Address

Aarhus
ECOLE POLYTECHNIQUE
France
Address
Route De Saclay
Palaiseau