Dataset Information

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 phosphorus 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 phosphorus 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.

SUBMITTER: Nygaard R

PROVIDER: S-EPMC5897578 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells.

Nygaard Rie R Romaniuk Joseph A H JAH Rice David M DM Cegelski Lynette L

The journal of physical chemistry. B 20170929 40

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 <sup>15</sup>N-labeled E. coli ribosomes. The experimental REDOR dephasing of shift-resolved lysyl amine nitrogens by phosphorus was comparable to that expected from a calculation of N-P distances involving the lysines included in the crystal structure coordinates. Among the nitrogen con ...[more]

PMID: 28901760

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Project description:The solution conformation behavior of a dermatan-derived tetrasaccharide--Delta HexA-(1-->3)-GalNAc4S-beta-(1-->4)-IdoA-alpha-(1-->3)-red-GalNAc4S (S is a sulfate group)--has been explored by means of NMR spectroscopy, especially by NOE-based conformational analysis. The tetrasaccharide was present as four species, two of which are chemically different in the anomeric orientation of the reducing 2-deoxy-2-acetamido-galactose (red-GalNAc) residue, while the other two are the result of different conformations of the iduronic acid (IdoA) unit. The two alpha-beta-interconverting anomers were present in a 0.6:1 ratio. Ring conformations have been defined by analysis of (3)J(H,H) coupling constants and interresidual NOE contacts. Both 2-deoxy-2-acetamido-galactose (GalNAc) residues were found in the (4)C(1) chair conformation, the unsaturated uronic acid (Delta-Hex A) adopts a strongly predominant half-chair (1)H(2) conformation, while the IdoA residue exists either in the (1)C(4) chair or in the (2)S(0) skewed boat geometries, in a 4:1 ratio. There is a moderate flexibility of Phi and Psi torsions as suggested by nuclear Overhauser effects (NOEs), molecular modeling (MM), and molecular dynamics (MD) studies. This was further investigated by residual dipolar couplings (RDCs). One-bond C--H RDCs ((1)D(C,H)) and long-range H-H ((3)D(H,H)) RDCs were measured for the tetrasaccharide in a phage solution and interpreted in combination with restrained MD simulation. The RDC-derived data substantially confirmed the validity of the conformer distribution resulting from the NOE-derived simulations, but allowed an improved definition of the conformational behavior of the oligosaccharides in solution. In summary, the data show a moderate flexibility of the four tetrasaccharide species at the central glycosidic linkage. Differences in the shapes of species with the IdoA in skew and in chair conformations and in the distribution of the sulfate groups have also been highlighted.

Dataset Information

Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells.

Publications

Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells.

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