The oncogenic GTP-binding P21 proteins encoded by ras- oncogenes, cause malignant transformation of cells in culture. They differ from the normal P21 protein coded for by Proto- oncogenes involved in regulation of the cell cycle in normal eukaryotic cells only in that they have single arbitrary amino acid substitutions at one critical position such as at 12, 13, 59, 61 or 63. This fact suggests that these substitutions on P21 peptide structure be investigated. A computer program based on the program ECEPP (Empirical Conformational Energies of Polypeptides and Proteins) will be used to calculate the low energy structures for normal P21 peptide segments and the same segments with malignancy-causing amino acid substitutions. The pattern of structural changes induced by these substitutions will then be used to predict which other amino acid substitutions may cause transformation of cells and which ones promote normal protein structures. These predictions can then be tested in experiments in which constructed genes containing these substitutions will be assayed for their abilities to transform NIH 3T3 cells in culture. The peptide segments to be investigated are the GTP-binding peptide Tyr 4-Ile 21 containing the critical residues 12 and 13 the peptide I1e 55-Met 67 containing the critical residues 59, 61 and 63. The solution structure of the synthetic GTP-binding 4-21 segments will be determined using high resolution NMR. The calculations will also be applied to determination of the preferred structures of the carboxyl terminal decapeptide, 180-189, known to be critical in membrane binding without which the protein is non-functional. Membrane binding depends on the presence of Cys 186 and the structural integrity of the 10 residues at the carboxyl terminal end of the protein. The results of the conformational analysis will be employed to make testable predictions as to amino acid substitutions that will result in loss of membrane binding and transforming activity.
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