Conformational transitions of polypeptide chain elongation factor Tu. I. Studies with hydrophobic probes.
The conformational difference between polypeptide chain elongation factor Tu (EF-Tu)-GTP and EF-Tu-GDP has been studied using hydrophobic and fluorescent probes. The interaction of EF-Tu-GDP with 1-anilino-8-naphthalenesulfonate (ANS) was measured in terms of the enhancement of the fluorescence intensity at the emission maximum of 475 nm. When EF-Tu-GDP-ANS complex was converted to EF-Tu-GTP-ANS complex by incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], there was a roughly 2-fold increase in fluorescence intensity and a blue shift of the emission maximum from 475 to 467 nm, indicating a conformational transition of the protein. The conformational change was found to be reversible and the spectrum promptly returned to that of EF-Tu-GDP-ANS complex upon addition of excess GDP. A similar change in the spectrum was also observed when aminoacyl-tRNA, but not deacylated tRNA, was added to EF-Tu-GDP-ANS complex. Measurement of the number of binding sites by gel filtration indicated that EF-Tu-GTP and EF-Tu-GDP bind 2.9 and 1.7 molecules of ANS, respectively. These results suggest that in EF-Tu-GTP the conformation was altered and one additional binding site for ANS was created at or near the site interacting with aminoacyl-tRNA. Another reagent, N-(1-anilinonaphthyl-4) maleimide (ANM) was covalently bound to the sulfhydryl group in EF-Tu-GDP which is essential for interaction with aminoacyl-tRNA. The binding could be determined spectrofluorometrically, since the reagent, which is nonfluorescent in aqueous solution, emitted a strong fluorescence upon binding with the sulfhydryl group, indicating a marked hydrophobicity of the local environment. Measurements of the kinetics of the binding revealed that ANM reacted rapidly with the sulfhydryl group in EF-Tu-GTP, while the reaction with that in EF-Tu-GDP proceeded more sluggishly. The difference in the reactivity of the sulfhydryl group essential for aminoacyl-tRNA binding between EF-Tu-GTP and EF-Tu-GDP probably reflects a conformational transition of the protein near the active site. These results, together with those on spin-label studies previously published (Arai, Kawakita, Kaziro, Maeda, & Onishi (1974) J. Biol. Chem. 249, 3311), demonstrate that reversible conformational transition does occur in EF-Tu on changing the ligand from GDP to GTP.
D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D004926:Escherichia coli; D006150:Guanine Nucleotides; D006160:Guanosine Triphosphate; D008301:Maleimides; D009281:Naphthalenes; D010442:Peptide Chain Initiation, Translational; D010445:Peptide Elongation Factors; D010649:Phenylalanine; D011485:Protein Binding; D011487:Protein Conformation; D012343:RNA, Transfer; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds
D000817:Anilino Naphthalenesulfonates; D006150:Guanine Nucleotides; D008301:Maleimides; D009281:Naphthalenes; D010445:Peptide Elongation Factors; D013438:Sulfhydryl Compounds; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D012343:RNA, Transfer