The ras-oncogene-encoded p21 proteins, known to be critical in regulation of the cell cycle, are activated by binding GTP and/or by substitutions of amino acids at critical positions such as at Gly 12 and Gln 61. The latter substitutions cause the protein to become oncogenic and to undergo significant conformational changes. Conformational analysis (based on the program ECEPP) in which the low energy (hence observed) conformations of residues 4-18 and 55-67 for the """"""""normal"""""""" and substituted peptides were computed suggested that oncogenic substitutions cause significant local conformational changes in the peptides. The effects of these local conformational changes on the structure of the whole protein will be assessed by generating them into recent x-ray crystallographic structures for the p21 protein and minimizing the energy of the resulting structures using a new method to avoid local energy minimum """"""""traps"""""""". The purpose is to determine which region(s) of the protein undergo major structural changes in the presence of GDP and GTP. These calculations have already been performed on the Val 12-containing p21 protein. It was found that the Gln 61 region and the region from residues 32-47 known to bind to a putative target protein GTPase activating protein (GAP) undergo major conformational changes. These results have been corroborated in recent x- ray crystal structures. The significance of the conformational changes in the p21 protein will be related to cell transformation by identifying possible effector regions of the protein that may be involved in interacting with intracellular """"""""target"""""""" proteins by introducing a photoaffinity-labelled p21 protein into NIH 3T3 cells using red cell fusion. Proteins with which p21 may interact can be covalently labelled, isolated and identified by immunoprecipitation, SDS-PAGE and microsequencing. The regions of the p21 protein that are involved in the interactions can be identified by peptide mapping and amino acid analysis of the photoaffinity-labelled protein. The computed effects of the conformational changes in the protein on these identified regions will be explored. Possible amino acid substitutions that would block computed critical conformational changes in effector-binding segments will be determined and the prediction tested in site-specific mutagenesis and microinjection experiments. In addition, peptides from the putative effector regions of p21 will be synthesized and tested for their ability to block ras-induced cell transformation.

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National Cancer Institute (NCI)
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Chemical Pathology Study Section (CPA)
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Upstate Medical University
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