Title.
Investigation of the interactions between remote amino acid residues in proteins using the directed evolution techniques (Prof. DR. Stanislav Zagrebelny)
The main goal of the Project:
The existing arsenal of computer analysis and modeling of the protein structures despite of many marked achievements during the last years still doesn’t allow to predict the influence of the amino acid substitutions at positions far away from catalytic centers or sites of ligand binding on the functional topography of protein molecule.
During the last decade the new branch of protein engineering has been formed, based on protein modification by means of random PCR mutagenesis of their genes which allows to obtain the variants of enzymes with distinct properties (substrate specificity, thermo stability, stability towards organic solvents, capability to “proper’ folding etc.). The analysis of the primary structure of the modified genes made it possible to evaluate the amino acid substitutions in protein variants obtained which are in correlation with change of the properties of the proteins and these changes are located in positions remote from functional sites. These facts observed but still remain to be considered systematically.
In terms of the Project the following investigations are planned:
- To obtain the modified variants of the proteins desired by means of some sequential rounds of random PCR mutagenesis;
- Selection of variants with modified functional properties;
- Creation of the library of modified variants;
- Investigation of their properties (catalytic activity, antigenic specificity etc.);
- Sequencing the modified variants and evaluation of the amino acid substitutions accompanying the functional differences between the variants obtained at the various steps of directed evolution;
- Construction of the structural models of the modified proteins;
- Formulation of hypotheses about the roles of interactions of remote amino acid residues in the functional properties of the protein molecule.
The first protein to be investigated is glucose isomerase – highly commercially attractive enzyme widely used in high fructose syrups production. Next step of the research will be performed on other enzymes and antigenic proteins of pathogenic microorganisms.