Ultra-high resolution 17O solid-state NMR spectroscopy of biomolecules: a comprehensive spectral analysis of monosodium L-glutamate·monohydrate.

Monosodium L-glutamate monohydrate, a multiple oxygen site (eight) compound, is used to demonstrate that a combination of high-resolution solid-state NMR spectroscopic techniques opens up new possibilities for (17)O as a nuclear probe of biomolecules. Eight oxygen sites have been resolved by double rotation (DOR) and multiple quantum (MQ) NMR experiments, despite the (17)O chemical shifts lying within a narrow shift range of <50 ppm. (17)O DOR NMR not only provides high sensitivity and spectral resolution, but also allows a complete set of the NMR parameters (chemical shift anisotropy and electric-field gradient) to be determined from the DOR spinning-sideband manifold. These (17)O NMR parameters provide an important multi-parameter comparison with the results from the quantum chemical NMR calculations, and enable unambiguous oxygen-site assignment and allow the hydrogen positions to be refined in the crystal lattice. The difference in sensitivity between DOR and MQ NMR experiments of oxygen in bio/organic molecules is also discussed. The data presented here clearly illustrates that a high resolution (17)O solid-state NMR methodology is now available for the study of biomolecules, offering new opportunities for resolving structural information and hence new molecular insights.

The determination of 17O NMR parameters of hydroxyl oxygen: a combined deuteration and DOR approach.

The direct detection of hydroxyl oxygen (O-H) by (17)O double-rotation (DOR) NMR is very challenging because of the strong O-H dipole interaction. It is shown that deuteration of the hydroxyl site overcomes this using glycine.HCl as an illustration. Two well-separated sets of narrow (linewidth approximately 80-100 Hz) resonances with their spinning-sidebands are observed for the carboxyl and hydroxyl oxygens in the DOR spectrum of [(17)O,(2)H]glycine.HCl. The chemical shift anisotropy of these sites is obtained from a simulation of the DOR spinning-sideband intensities. The chemical shift span (Omega) for the carboxyl oxygen is found to be much larger than that of the hydroxyl oxygen, with Omega values of 540 +/- 15 and 210 +/- 10 ppm, respectively.

High-resolution 17O double-rotation NMR characterization of ring and non-ring oxygen in vitreous B2O3.

The application of double rotation (DOR) NMR to crystalline materials (both inorganic and organic) has made tremendous strides in providing site-specific information about materials in recent years. However (17)O DOR has yet to demonstrate its potential in disordered materials such as glasses. In the present study, we have successfully recorded high resolution (17)O DOR spectra of vitreous B(2)O(3) (v-B(2)O(3)), a highly effective glass-forming oxide of considerable technological importance. Two distinct oxygen sites are resolved and a complete set of (17)O NMR parameters were determined from the DOR spectra. These were assigned to oxygen atoms in the planar boroxol ring [B(3)O(6)] and in the non-boroxol [BO(3)] groups which share oxygen with the ring boron atoms. This assignment was based on the similarity of all of their (17)O parameters with those found by DFT calculation for caesium enneaborate, Cs(2)O.9B(2)O(3), which has two boroxol rings in its structure. The boroxol ring oxygens have a more positive chemical shift, a larger shift anisotropy and a smaller electric field gradient than non ring oxygens (O(R): delta(iso) = 100 +/- 1 ppm, span = 180 +/- 20 ppm, skew = -0.4 +/- 0.1, P(q) = 5.0 +/- 0.2 MHz; O(NR): delta(iso) = 86 +/- 1 ppm, span = 100 +/- 20 ppm, skew = 0.1 +/- 0.1, P(q) = 5.7 +/- 0.2 MHz). The relative proportions of the two sites in v-B(2)O(3) are approximately 1 : 1, as expected if all three boron atoms in the boroxol ring are each connected to one oxygen in a linking [BO(3)] group and there are very few [BO(3)]-[BO(3)] linkages. We see no evidence for a third oxygen site such as has been reported in an earlier study of v-B(2)O(3). This work demonstrates the potential of (17)O DOR to provide site-specific information in disordered materials.

Separation of isotropic chemical and second-order quadrupolar shifts by multiple-quantum double rotation NMR.

Using a two-dimensional multiple-quantum (MQ) double rotation (DOR) experiment the contributions of the chemical shift and quadrupolar interaction to isotropic resonance shifts can be completely separated. Spectra were acquired using a three-pulse triple-quantum z-filtered pulse sequence and subsequently sheared along both the nu(1) and nu(2) dimensions. The application of this method is demonstrated for both crystalline (RbNO(3)) and amorphous samples (vitreous B(2)O(3)). The existence of the two rubidium isotopes ((85)Rb and (87)Rb) allows comparison of results for two nuclei with different spins (I=3/2 and 5/2), as well as different dipole and quadrupole moments in a single chemical compound. Being only limited by homogeneous line broadening and sample crystallinity, linewidths of approximately 0.1 and 0.2 ppm can be measured for (87)Rb in the quadrupolar and chemical shift dimensions, enabling highly accurate determination of the isotropic chemical shift and the quadrupolar product, P(Q). For vitreous B(2)O(3), the use of MQDOR allows the chemical shift and electric field gradient distributions to be directly determined-information that is difficult to obtain otherwise due to the presence of second-order quadrupolar broadening.

New limits for solid-state 17O NMR spectroscopy: complete resolution of multiple oxygen sites in a simple biomolecule.

A solid-state 17O NMR 1H-decoupled double angle rotation (DOR) study of monosodium l-glutamate monohydrate (l-MSG) is reported. It is shown that all eight inequivalent sites can be resolved with DOR line widths ( approximately 65 Hz) approximately 120 times narrower than those in the MAS spectrum. The lines are tentatively assigned on the basis of their behavior under proton decoupling and the isotropic chemical shift and the quadrupole interaction parameter for each extracted by a combination of DOR and 3Q MAS at variable magnetic fields. With a shift range of approximately 45 ppm for these similar oxygen sites and spectral resolution under DOR comparable to that for spin-1/2 nuclei, solid-state 17O NMR should have tremendous potential in the study of biomolecules.

Solid-state 17O NMR as a probe for structural studies of proteins in biomembranes.

We report the first example of 17O NMR spectra from a selectively labeled transmembrane peptide, 17O-[Ala12]-WALP23, as a lyophilized powder and incorporated in hydrated phospholipid vesicles. It is shown that at high magnetic field it is feasible to apply 17O NMR to the study of membrane-incorporated peptides. Furthermore, we were able to estimate distances within the selectively labeled WALP peptide, which represents a consensus transmembrane protein sequence. This work opens up new applications of 17O solid-state NMR on biological systems.