Objective
THIS WILL BE A MODEL SYSTEM FOR DEVELOPMENT OF RATIONAL APPROACHES FOR PROTEIN ENGINEERING BY COMBINED GENETIC, STRUCTURAL AND MODELLING TECHNIQUES. THE ADVANTAGE OF BARNASE IS THAT IT IS SUFFICIENTLY SMALL TO BE READILY STUDIED BY NMR AND X-RAY CRYSTALLOGRAPHY. IT CAN BE HOPED THAT A DETAILED STUDY OF THIS PROTEIN WILL LEAD TO THE ELUCIDATION OF RULES GOVERNING PROTEIN FOLDING. THE KNOWLEDGE OF THESE RULES IS A MAJOR BOTTLENECK IN DESIGNING NOVEL PROTEINS.
Research was carried out in order to develop rational approaches for engineering modified protein using genetic engineering techniques. Sequence modifications were made in barnase (a small ribonuclease from Bacillus amyloliquefaciens) to probe the catalytic mechanism in the ribonucleases about which appreciable controversy persists and to dissect the contributions from hydrophobic and electrostatic interactions to protein stability. Mutations were also used as reporter groups in kinetic studies of protein folding to provide information on the conformation of the transition state for unfolding. Crystallographic analysis was performed on the enzyme complex with a deoxydinucleotide inhibitor (dGpC). Molecular modelling was used to help interpret data on protein nucleic acid interactions and catalysis, and contributions of electrostatic and hydrophobic interactions to protein stability were analysed by computer simulations. Interactions of barnase with its intracellular protein inhibitor, barstar, were studied via random mutagenesis of the barstar gene.
Parameters of the enzyme catalyzed reaction were determined for short nucleotides and ribonucleic acid (RNA) substrates, and shown to be significantly different. Residues of barnase responsible for catalysis were established. The structure of the complex of barnase with a deoxydinucleotide inhibitor was solved. It shows an unproductive binding mode for the dinucleotide involving enzyme subsites that are different from the primary recognition site for guanine in related ribonucleases. Based on these results, molecular modelling was used to study the interaction of the enzyme with RNA analogues, and to rationalize the catalytic mechanism. Appreciable progress was made in understanding how hydrophobic and electrostatic interactions contribute to protein stability and on the physical origins of the observed effects. An arginine and a cysteine residue in barstar (respectively in positions 75 and 40) were both identified as participating in the barnase barstar interaction.
THE MAIN OBJECTIVE OF THIS PROJECT WILL BE TO INTRODUCE MODIFICATIONS (AMINO ACID SUBSTITUTIONS) IN THE ACTIVE SITE OF BARNASE (I.E. BACILLUS AMYLOLIQUEFACIENS EXTRACELLULAR RIBONUCLEASE) SO AS TO STUDY AND ALTER THE SUBSTRATE SPECIFICITY AS WELL AS OTHER ENZYMATIC PROPERTIES. THE FIRST STEP WILL BE TO PRODUCE MODIFIED BARNASE AND BARSTAR (THE INTRACELLULAR INHIBITOR OF BARNASE). CERTAIN RESIDUES, KNOWN TO BE INVOLVED IN CATALYSIS, WILL BE SUBSTITUTED AND THEIR ROLE IN THE CATALYTIC PROCESS WILL BE INVESTIGATED. MUTAGENESIS WILL ALSO BE USED TO IDENTIFY RESIDUES INVOLVED IN SUBSTRATE BINDING, AS WELL AS TO INVESTIGATE THE INTERACTION BETWEEN BARNASE AND BARSTAR.
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 biochemistry biomolecules nucleic acids
- natural sciences biological sciences biochemistry biomolecules proteins protein folding
- natural sciences chemical sciences catalysis
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
- natural sciences mathematics applied mathematics mathematical model
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Coordinator
1040 BRUSSELS
Belgium
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