Js. Hu et Ag. Redfield, CONFORMATIONAL AND DYNAMIC DIFFERENCES BETWEEN N-RAS P21 BOUND TO GTP-GAMMA-S AND TO GMPPNP AS STUDIED BY NMR, Biochemistry, 36(16), 1997, pp. 5045-5052
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.