Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET

Abstract

Thiouridine at position 8 (s4U8) of tRNAf1Met was spin-labeled with the nitroxide free radical, N-(1-oxyl-2,2,5,5-Tetramethyl-3-pyrrolidinyl) bromacetamide, for proton nuclear magnetic resonance spectroscopic studies. The well-resolved methyl peak of ribothymidine is unperturbed, but the peak tentatively assigned to the C-5 methylene group of dihydrouridine is considerably broadened in spin-labeled tRNAf1Met. Of the approximately 27 slowly exchanging protons observed in the region between 11 and 15 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid, the equivalent of about five protons apparently disappeared in spin-labeled tRNAf1Met. The well-resolved single proton at 14.8 ppm was missing not only in the paramagnetic species, but also in the diamagnetic reduced form of spin-labeled tRNAf1Met, and was unequivocally identified as a hydrogen bond involving s4U8 by comparison of several forms of tRNAf1Met specifically modified at s4U. Evidence that the perturbation of a second single proton resonance at 14.6 ppm (shift and broadening) is coupled to the loss of a tertiary hydrogen bond involving residue 8, arises from the same modified forms. The resolved resonances in the methyl and N-H regions, particularly the resonance at 14.6 ppm as well as the four N-bonded proton resonances at higher field which are broadened solely due to their proximity to the unpaired electron of the spin label, provide specific indicators of the geometry of tRNAf1Met structure in solution. Their observability by nuclear magnetic resonance spectroscopy opens up the possibility of monitoring distance changes among the base residues of spin-labeled tRNAf1Met upon its interaction with aminoacyl-tRNA synthetase and other enzymes.