Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells

Abstract

Solid-state NMR is a powerful tool for quantifying chemical composition and structure in complex assemblies and even whole cells. We employed N{P} REDOR NMR to obtain atomic-level distance propensities in intact 15N-labeled E. coli ribosomes. The experimental REDOR dephasing of shift-resolved lysyl amine nitrogens by phosphorous was comparable to that expected from a calculation of N-P distances involving the lysines included in the crystal structure coordinates. Among the nitrogen contributions to the REDOR spectra, the strongest dephasing emerged from the dipolar couplings to phosphorous involving nitrogen peaks ascribed primarily to rRNA and the weakest dephasing arose from protein amide nitrogens. This approach is applicable to any macromolecular system and provides quantitative comparisons of distance proximities between shift-resolved nuclei of one type and heteronuclear dephasing spins. Enhanced molecular specificity could be achieved through the use of spectroscopic filters or specific labeling. Furthermore, ribosome 13C and 15N CPMAS spectra were compared with those of whole cells from which the ribosomes were isolated. Whole-cell signatures of ribosomes were identified and should be of value in comparing overall cellular ribosome content in whole-cell samples.

Spectral comparisons of mammalian cells and intact organelles by solid-state NMR

“…The E. coli whole cell spectrum, however, is enhanced in the peaks between 35–40 ppm, 60–100 ppm and 135–160 ppm in the comparison as normalized to the carbonyls peaks. These peaks correspond to the major carbon signatures present in E. coli ribosomes (Nygaard et al 2017) and is consistent with the increased percentage of the cell volume occupied by ribosomes in E. coli , lacking other organelles, as compared to HeLa cells. In contrast, the peaks at 130 ppm and 30 ppm are enriched in the HeLa cell when compared with the E. coli spectrum, and these peaks were notably enhanced in the isolated mitochondria spectrum.…”

“…Ribosomes are not membrane-bound structures, but are the cellular machines that carry out protein synthesis. We recently reported the 13 C and 15 N CPMAS spectra of intact ribosomes isolated from E. coli (Nygaard et al, 2017). Ribosomes naturally exhibit dramatic differences from whole cells and other cellular assemblies, based on their unique protein-RNA composition and the defining 13 C chemical shifts for nucleic acid carbons.…”

“…Moreover, this approach had the sensitivity to distinguish cells as being treated with the antibiotic fosfomycin (a cell-wall inhibitor) and chloramphenicol (a protein synthesis inhibitor) (Nygaard et al, 2015). More recently, we reported the 13 C CPMAS spectrum of intact bacterial ribosomes which can account for up to one-fourth of the cell mass and could help reveal compositional changes in whole cells that impact ribosome production (Nygaard et al, 2017).…”

In-Cell Quantification of Drugs by Magic-Angle Spinning Dynamic Nuclear Polarization NMR

“…In the spectrum, resonances arising from the cellular background are clearly observable between 100 and 120 ppm (assignable mostly to protein amide backbone resonances and tryptophan, asparagine, and glutamine side chains) and between 80 and 100 ppm (assignable mostly to arginine side chains and nucleic acids). 39 Resonances from the histidine standard are observable at ∼40 ppm (amine group) and ∼170 and ∼250 ppm (aromatics). The presence of two distinct resonances for the aromatics indicates that the pH of the suspended solution is probably close to neutral.…”

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation

“…X-ray crystallography has provided high-resolution views of translational components, including ribosomes. NMR spectroscopy is used to determine structural dynamics of ribosomal domains (8) and nascent protein chains (9), and recently whole ribosomes using solid state methods (10–13), but is hindered by fundamental limitations in solution for large (>500 kDa) systems. Recent advances in cryogenic electron microscopy (cryoEM) have yielded much excitement in the field of translation by providing high-resolution information about translational intermediates and larger complexes that have been previously inaccessible by other structural methods.…”