GUANOSINE TRIPHOSPHATE BINDING OF RAS PROTEIN BY NMR AND CD SPECTROSCOPY

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The objective of this project is to study the chemical interactions of the ras proteins (p21) and guanosine nucleotides. The guanosine triphosphate (GTP) and guanosine diphosphate (GDP) binding site of these proteins are to be precisely determined by nuclear magnetic resonance (NMR), circular dichroism (CD), equilibrium dialysis, synthesis of peptides by the solid-phase method, and calculations of binding constants and therymodynamic parameters of GTP-peptide complexes. At present, the research is focused on the following areas: (1) The solid-phase synthesis of the 34 amino acid residue N-terminal sequence of both the protooncogene and the oncogene of ras proteins (p21). (2) Purification of peptides by column chromatography and high performance liquid chromatogphy (HPLC). (3) Studies on the conformational difference between the glycine-containing (Glypeptide) and aline-containing (Val-peptide) 34 amino acid residue-peptide by CD spectroscopy (the glycine or valine is at position 11). (4) Studies on the conformational changes of both Gly-peptide and Val-peptide upon addition of either formational changes of both Gly-peptide and Val-peptide upon addition of either GTP or GDP using both NMR and CD spectroscopy. (5) Studies on the interaction between peptides and GTP by NMR spectroscopy. Results obtained so far are as follows: (1) The information obtained from CD studies shows that both Gly-peptide and Val-peptide adopted predominately beta-sheet conformation (55-60%) in a nonionic detergent solution. (2) Upon addition of GTP to the peptide solution, both Gly-peptide and val-peptide conformation remained in the beta-sheet conformation. However, the ramdom coil conformation gradually increases at the expense of beta sheet conformation. (3) When the Glky-peptide was added to the GTP solution containing sodium dodecyl sulfate, the line widths of P-31 signals of both beta and gamma resonances of GTP were broadened significantly (about 10 Hz), implying that binding probably occurs between GTP and the Gly-peptide.