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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 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 BATTLENECK WHICH MAKES IMPOSSIBLE THE DESIGN OF NOVEL PROTEINS.
The ribonuclease excreted by Bacillus amyloliquefaciens, Barnase, was cocrystallised with the deoxydinucleotide d(GpC). The crystal structure was determined by molecular replacement from a model of free Barnase previously derived by Mauguen et al. Refinement was carried out using data to 1.9 angstroms resolution. The final model, which has a crystallographic R factor of 22%, includes 869 protein atoms, 38 atoms from d(GpC), a sulphate ion and 73 water molecules. Only minor differences from free Barnase are seen in the protein moiety, the root mean square C-alpha movement being 0.45 angstroms. The dinucleotide has a folded conformation. It is located near the active site of the enzyme, but outside the protein molecule and making crystal packing contacts with neighbouring molecules. The guanine base is stacked on the imidazole ring of active site His102, rather than binding to the so called recognition loop as it does in other complexes of guanine
nucleotides with microbial nucleases. The deoxyguanosine is syn, with the sugar ring in C-2'-endo conformation; the deoxycytidine is anti and C-4'-exo. In addition to the stacking interaction, His102 hydrogen bonds to the free 5' hydroxyl, which is located near the position with the 3' phosphate group is found in other inhibitors of microbial ribonucleases. While the mode of binding observed with d(GpC) and Barnase would be nonproductive for a dinucleotide substrate, it may define a site for the nucleotide product on the 3' side of the hydrolysed bond.

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.
ESTABLISHMENT OF THE THREE DIMENTIONAL STRUCTURE OF DIFFERENT FORMS OF CRYSTALLINE BARNASE, A SMALL BACTERIAL RIBONUCLEASE.
THE DIFFRACTION PATTERN WILL BE COLLECTED TO HIGH RESOLUTION ON A FOUR-CIRCLE DIFFRACTOMETER OR ON THE SYNCHROTRON RADIATION SET-UP AT LURE (ORSAY).
THE FOLLOWING CRYSTALS WILL BE ANALYSED SEQUENTIALLY :
1- NATIVE BARNASE COMPLEXES WITH NUCLEOTIDE SUBSTRATES OR INHIBITORS.
2- MUTANT BARNASE OBTAINED BY GENETIC ENGINEERING.
3- NATIVE BARNASE COMPLEXED WITH ITS NATURAL INHIBITOR " BARSTAR ".

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Université de Paris XI (Université Paris-Sud)
Address

91405 Orsay
France

Participants (3)

IMPERIAL COLLEGE
United Kingdom
Address
South Kensington Campus
SW7 2AZ London
PLANT GENETIC SYSTEMS NV
Belgium
Address

Gent
ULB
Belgium
Address

Bruxelles