Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes

15N-(1H) NMR spectroscopy has been employed to investigate the dynamic properties of uniformly 15N-labeled Escherichia coli ribosomes. Despite the large molecular weight of the ribosome (~2.3 MDa), [(1H-15N)] heteronuclear single-quantum correlation (HSQC) spectra reveal approximately 100 well-resolved resonances, most of which originate from two of the four C-terminal domains of the stalk proteins L7/L12. Heteronuclear pulse-field gradient NMR experiments confirm that these resonances correspond to components diffusing at a rate consistent with the intact ribosome.
Measurements of 15N longitudinal relaxation times (T₁) and spin-lock relaxation times (T₁ρ) indicate that the observed domains exhibit anisotropic tumbling. Their apparent rotational correlation times are GSK429286A significantly longer than expected for free L7/L12 domains, yet much shorter than would be anticipated for domains rigidly embedded within the ribosome, suggesting partial mobility. These relaxation data enable orientation of the C-terminal domains relative to the ribosome’s rotational diffusion tensor.
Upon binding of elongation factor G (EF-G), the resonances from L7/L12 disappear, indicating a marked loss of mobility. This observation aligns with cryo-electron microscopy studies showing that the ribosomal stalk adopts a fixed orientation upon EF-G binding. In addition to revealing the dynamic behavior of L7/L12, these findings demonstrate the effectiveness of heteronuclear NMR in characterizing flexible regions within large macromolecular assemblies and lay the groundwork for future NMR investigations of functional ribosomal complexes during protein synthesis.