CONFORMATIONAL AND DYNAMIC DIFFERENCES BETWEEN N-RAS P21 BOUND TO GTP-GAMMA-S AND TO GMPPNP AS STUDIED BY NMR

Heteronuclear-edited proton-detected NMR methods are used to study the nucleotide-dependent conformational changes between the GMPPNP form o f human N-ras P21 as compared to GDP and GTP gamma S forms. Full-lengt h N-ras P21 was also compared with protein truncated beyond residue 16 7, to search for interaction points between the more invariant part of the protein and the variable C-terminal section. In both cases, the r eporter was the N-15-H 2D spectrum of aspartate amide groups labeled w ith N-15. Small truncation-induced changes were seen in the spectrum a t the resonances of Asp-54, -108, and -109 which are not far from the C-terminal and, surprisingly, at Asp-57 which is more remote. The spec trum obtained for the GMPPNP-ligated form is similar to that of the GT P gamma S form, except that peaks of several residues are weak at low temperature, and strongly temperature-dependent in their intensity, an d a new resonance appears at 15 degrees C and above. The observations are discussed in terms of a two-state model for the GMPPNP-ligated pro tein, previously proposed by Geyer et al.