NMR Studies of the Conformational Change in Human N-p21^ras Produced by Replacement of Bound GDP with the GTP Analog GTPǗS

¹H-Detected ¹⁵N-edited NMR in solution was used to study the conformational differences between the GDP-and GTPᵧS-bound forms of human N-p21^ras. The amide protons of ¹⁵N-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 ¹³C-labeled amino acids in addition to [¹⁵N]glycine and [¹⁵N]iosleucine and by replacing Mg²⁺ by Co²⁺. When GTP7S replaced GDP in the active site of p21^ras, 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 GTPᵧS 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 GTPᵧS. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the a-helix containing Ile-21 is also shown to be affected.