NMR-STUDY OF THE PHOSPHORYL BINDING LOOP IN PURINE NUCLEOTIDE PROTEINS - EVIDENCE FOR STRONG HYDROGEN-BONDING IN HUMAN N-RAS P21

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 β-phosphate oxygens of GDP. © 1990, American Chemical Society. All rights reserved.