CONFORMATIONAL DIFFERENCES BETWEEN COMPLEXES OF ELONGATION-FACTOR TU STUDIED BY F-19-NMR SPECTROSCOPY

An analogue of elongation factor Tu (EF-Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorotyrosine. The F-19-N MR spectra of the binary complexes of this protein with GDP, GT? and e longation factor Ts (EF-Ts) and the ternary complexes EF-Tu . GDP . au rodox and EF-Tu . GDP . EF-Ts were measured. EF-Tu contains ten tyrosi ne residues and all of the complexes studied gave complex F-19 spectra with overlapping resonances. EF-Tu . GDP gave a spectrum in which two signals were markedly different from those shown by the other complex es, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes. Such large d ownfield shifts can be explained by second-order electric field shield ing effects resulting from these two tyrosine residues being in a ster ically constrained environment in EF-Tu - GDP and with the steric rest raints being released in all of the other complexes. The X-ray diffrac tion structure of EF-Tu - GDP shows that Tyr87 in the N-terminal domai n (domain I) and Tyr309 in die C-terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF-Tu previously implicated in the structural chang es between EF-Tu . GDP and EF-Tu - GTP by other workers. If these tyro sine residues correspond to the two downfield resonances of the spectr a of EF-Tu . GDP, the results from the F-19-NMR would be consistent wi th these earlier indications that domain I interacts closely with doma in III in EF-Tu . GDP and that the amino acids between Gly83 and Gly10 0 are an important part of this interaction. For all the other complex es studied, these tyrosines are in a less sterically crowded environme nt consistent with a weaker interaction between die two domains. The F -19-NMR spectrum of the typsin-cleaved product of EF-Tu . GDP, from wh ich the X-ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleav age causes no large changes in the protein's structure.