Ribonucleases (RNases) are enzymes which constitute optimal models for the study processes such as catalysis, enzyme-substrate interaction and protein folding. Given the involvement of some members of this class of proteins on sereval important cell events new possibilities are now open to study. The present project aims to the advancement in the Knowledge of the molecular basis relating structure and function in the RNase family. Four different goals have been defined.From the methodological point of view, they have in common an analysis of the effects caused by the introduction, by sitedirected mutagenesis of specific aminoacid substitutions: 1) Study of the molecular and structural bases involved in the enzymatic activity present in the eosinophyl cationic protein (ECP), the eosinophyl-derived neurotoxin (EDN) and human pancreatic RNase (HP-RNase). These will be compared to those of bovine pancreatic RNase A; in each case the analyses will be specific for the particular protein used and will depend on all the available knowledge regarding the mechanism of catalysis and three-dimensional structure. 2) Study of the contribution of some critical residues from the hydrophobic core to the general stabailiaty and fording pathway of pancreatic RNases. The effect of pressure on the protein structure will beanalysed in relation to the effect of other denaturing agents; in this goal, RNase A will be the chosen model because of the amount of data already available on this subject. 3) Preparation of human pancreas RNase (HP-RNase) variants aiming at increasing its cytotpxicity. Conversely, variants of onconase will also be obtained with the aimof increasing its RNase activity; the choice of particular variant is based on the analysis of the three-dimensional structure of each native protein. Studies on the structure and thermostability of each variant will also be carried out. 4) Study of the molecular basis of the cytortoxicity of the eosinophyl-cationic protein
|Effective start/end date||19/12/00 → 19/12/03|
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