Involvement of histidine residues in the substrate binding of elongation factor Tu from Thermus thermophilus: Proton nuclear magnetic resonance and photooxidation study

Abstract

Proton nuclear magnetic resonance (nmr) spectra (270 MHz) were measured of polypeptide chain elongation factor Tu (EF-Tu) from an extreme thermophile, Thermus thermophilus. This protein was stable enough for a series of nmr measurements at temperature as high as 50 °C. For histidine C 2 protons, pH dependences of nmr chemical shifts were measured in the pH range from 5.5 to 8.0. The nmr titration curve of one histidine residue of free EF-Tu was markedly affected by the binding with GDP. This titration curve was further affected by the ligand substitution from GDP to GTP, indicating that this histidine is involved in the binding of EF-Tu with guanine nucleotides. The nmr titration curve of another histidine was also affected by the ligand substitution from GDP to GTP. The results of photooxidation experiments suggest that histidine residues are involved in the binding of EF-Tu with guanine nucleotides as well as with aminoacyl-tRNA and/or ribosomes.