Quadrupole Central Transition ¹⁷O NMR Spectroscopy of Biological Macromolecules in Aqueous Solution

Abstract

We demonstrate a general nuclear magnetic resonance (NMR) spectroscopic approach in obtaining high-resolution (17)O (spin-5/2) NMR spectra for biological macromolecules in aqueous solution. This approach, termed quadrupole central transition (QCT) NMR, is based on the multiexponential relaxation properties of half-integer quadrupolar nuclei in molecules undergoing slow isotropic tumbling motion. Under such a circumstance, Redfield's relaxation theory predicts that the central transition, m(I) = +1/2 ↔ -1/2, can exhibit relatively long transverse relaxation time constants, thus giving rise to relatively narrow spectral lines. Using three robust protein-ligand complexes of size ranging from 65 to 240 kDa, we have obtained (17)O QCT NMR spectra with unprecedented resolution, allowing the chemical environment around the targeted oxygen atoms to be directly probed for the first time. The new QCT approach increases the size limit of molecular systems previously attainable by solution (17)O NMR by nearly 3 orders of magnitude (1000-fold). We have also shown that, when both quadrupole and shielding anisotropy interactions are operative, (17)O QCT NMR spectra display an analogous transverse relaxation optimized spectroscopy type behavior in that the condition for optimal resolution depends on the applied magnetic field. We conclude that, with the currently available moderate and ultrahigh magnetic fields (14 T and higher), this (17)O QCT NMR approach is applicable to a wide variety of biological macromolecules. The new (17)O NMR parameters so obtained for biological molecules are complementary to those obtained from (1)H, (13)C, and (15)N NMR studies.

QUADRELAX: A Program for Computing Relaxation Data for Quadrupolar Nuclei, Including Chemical Exchange Effects

“…Furthermore, our approach to the problem is such that it could easily be extended to include any number of exchange sites more than two. QUADRELAX also duplicates the results of Jaccard et al's work (18) as well as previously mentioned references (1,5,(9)(10)(11)(12). According to Pople's highly cited article (17), the different components of a multiplet arose from a spin 1 2 nucleus coupled to a spin I nucleus have different line width.…”

“…<O >5TraceðOrÞ [1] in Eq. [1], the bracket sign stands for averaging of all possible results, which take place when the observable O is measured for a single subsystem, that is, a single spin, and the bar sign implies averaging on an ensemble of these single subsystems.…”

NMR spectroscopy up to 35.2 T using a series-connected hybrid magnet

“…The relaxation of spin > 1/2 nuclei from the quadrupolar interaction is multi-exponential and the behavior of the slow relaxing component is analogous to that of the central-transition of quadrupolar nuclei in solids. The quadrupole central-transition (QCT) line width in the slow tumbling regime has a strong field dependence, as demonstrated recently by the variable-field studies of 17 O-enriched oxalate bound to chicken ovotransferrin (total molecular weight ~ 80 kDa) [32, 33] and pyruvate kinase (240 kDa) [34] up to 21.1 T. Fig. 10 shows 17 O QCT spectra of [3,5,6- 17 O]-D-glucose in glycerol solution at different magnetic fields.…”

NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for Molecular Structural Biology

“…One approach that exploits quadrupolar relaxation and has been successful for the study of quadrupolar nuclei in the context of biological macromolecules (including 17 O, 27 Al, and 51 V) is quadrupole central transition (QCT) NMR (48, 158–160), which utilizes relaxation properties of the central transition at high magnetic fields (i.e. the slow tumbling regime) to obtain narrow lines.…”

“…the slow tumbling regime) to obtain narrow lines. The combination of high magnetic fields and narrow lines allows for the observation of fine structural details and applications to larger biological systems (158), as demonstrated in 17 O QCT experiments to study the catalytic mechanism of the enzyme Tryptophan synthase (48). …”