The triple isotopic substitution method in small angle neutron scattering. Application to the study of the ternary complex EF-Tu · GTP · aminoacyl-tRNA

Abstract

The TIS (triple isotopic substitution) method in small angle neutron scattering was applied to determine the radius of gyration of polypeptide elongation factor Tu (EF-Tu) from E. coli associated with GDP and within the ternary complex EF-Tu · GTP · aminoacyl-tRNA. The results showed that, within errors of about 1 Å, there is no change in the radius of gyration of the EF-Tu moiety upon ternary complex formation. Experiments were performed in H 2O buffer, in which complex formation could be followed on an absolute scale because of the relatively large contrast of both protein and tRNA. The TIS method is based on the analysis of a scattering curve that is the difference between the scattering of two solutions containing appropriately deuterium labelled particles. A necessary condition for the application of the method is that the two solutions are identical in all respects except for the extent of deuterium label. The main properties of TIS that make it very useful for the study of complex particles in solution were confirmed by this study. These are the elimination of interparticle effects in the difference curve, the ‘invisibility’ of unlabelled parts of the particles and the independence of the difference scattering curve on the buffer 2H 2OH 2O content. The last property is of particular interest for the study of interactions that may be influenced by 2H 2O, since, contrary to classical contrast variation methods, TIS experiments can be performed in H 2O buffer alone.