NMR study of the phosphoryl binding loop in purine nucleotide proteins: evidence for strong hydrogen bonding in human N-ras p21

Abstract

The structure of the phosphoryl binding region of human N-ras p21 was probed by using heteronuclear proton-observed NMR methods. Normal protein and a Gly-12----Asp-12 mutant protein were prepared with two amino acids labeled with 15N at their amide positions: valine and glycine, aspartic acid and glycine, and lysine and glycine. We completed the identification of amide 15NH resonances from Gly-12 and Asp-12 to the end of the phosphoryl binding domain consensus sequence (Lys-16) in protein complexed with GDP and have made tentative amide identifications from Val-9 to Ser-17. The methods used, together with initial identifications of the Gly-12 and -13 amide resonances, were described previously [Campbell-Burk, S. (1989) Biochemistry 28, 9478-9484]. The amide resonances of both Gly-13 and Lys-16 are shifted downfield below 10.4 ppm in both the normal and mutant proteins. These downfield shifts are presumed to be due to strong hydrogen bonds with the beta-phosphate oxygens of GDP.