Conformational transitions of polypeptide chain elongation factor Tu. I. Studies with hydrophobic probes.:I. Studies with Hydrophosbic 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 con-formational 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• GTP• 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.<br> 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 fluore-scence 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.<br> These results, together with those on spin-label studies previously published (Arai, Kawakita, Kaziro, Maeda, & Ohnishi (1974) J. Biol. Chem. 249, 3311), demon-strate that reversible conformational transition does occur in EF-Tu on changing the ligand from GDP to GTP.