Neutron scattering study of the binding of tRNAPhe to Escherichia coli phenylalanyl-tRNA synthetase

Abstract

E. coli phenylalanyl-tRNA synthetase was characterized by small-angle neutron scattering. In solution (20 mM imidazole hydrochloride, pH 7.6, 10 mM 2-mercaptoethanol and 0.1 mM EDTA), this enzyme has a MW of 227 K [kilodaltons] .+-. 20 K with a radius of gyration (RG) of 48.3 .+-. 0.6 .ANG., independent of the presence of MgCl2 up to 50 mM. The change of the scattering upon adding tRNAPhe to the enzyme has been followed with 10 mM MgCl2 present in the buffer. One enzyme molecule is capable of binding 2 tRNAPhe molecules with affinity constants > 106 M-1. Parallel titration experiments in 73% 2H2O, close to the matching point of tRNA, show that the RG of the enzyme is not changed by the binding of 1 or 2 tRNAPhe molecules. These results are compared with quasi-electric light scattering studies where the addition of MgCl2 or tRNAPhe caused dramatic changes of the apparent translational diffusion constant of phenylalanyl-tRNA synthetase.