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Content archived on 2024-04-15

ENGINEERING OF AN EXTRACELLULAR RIBONUCLEASE BY GENE MODIFICATION

Objective

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 N.M.R. 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 WHICH MAKES IMPOSSIBLE THE DESIGN OF NOVEL PROTEIN.
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.
INTERACTIVE COMPUTER GRAPHICS AND COMPUTER SIMULATIONS WILL BE APPLIED TO ANALYSE THE KNOWN CRYSTAL STRUCTURE OF THE NATIVE BARNASE PROTEIN AND TO ASSESS THE EFFECTS OF AMINO-ACID SUBSTITUTIONS ON THE NATIVE CONFORMATION AND ON ITS DYNAMIC PROPERTIES.
SUCH STUDIES, CARRIED OUT WITH A 32-BIT MINI COMPUTER, AN INTERACTIVE COLOUR GRAPHICS PS300/FROM EVANS AND SUTHERLAND AND A POWERFUL ARRAY PROCESSOR, WILL BE PERFORMED WITH THE AIM OF DEVELOPING PREDICTIVE METHODS FOR ENZYME-SUBSTRATE AND ENZYME-EXHIBITOR INTERACTIONS.

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UNIVERSITE LIBRE DE BRUXELLES
EU contribution
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Address
UNITE DE CONFORMATION DES MACROMOLECULES BIOLOGIQUES AVENUE PAUL HEGER P2
1050 BRUSSELS
Belgium

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Participants (3)