NMR-STUDIES OF THE CONFORMATIONAL CHANGE IN HUMAN N-P21RAS PRODUCED BY REPLACEMENT OF BOUND GDP WITH THE GTP ANALOG GTP-GAMMA-S

H-1-Detected N-15-edited NMR in solution was used to study the conformational differences between the GDP- and GTPgammaS-bound forms of human N-p21ras. The amide protons of N-15-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various C-13-labeled amino acids in addition to [N-15]glycine and [N-15]iosleucine and by replacing Mg2+ by Co2+. When GTPgammaS replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTPgammaS for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTPgammaS. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.