New Limits for Solid-State ¹⁷O NMR Spectroscopy:  Complete Resolution of Multiple Oxygen Sites in a Simple Biomolecule

Abstract

Nuclear magnetic resonance (NMR) is a key technique in providing atomic scale information on molecular architecture. Solidstate NMR approaches are playing an increasing role in biomolecular science; however, almost all such NMR reports concern spin-1/2 nuclei ( 1 H, 13 C, 15 N). Oxygen is one of the most important and abundant elements in biological systems, 1 but it is little studied by NMR. Since oxygen plays a central role in many biological interactions, such as protein-protein, metal-protein, and in nucleic acids, it would be beneficial to be able to study the oxygen directly. From the NMR point of view, spin-1/2 nuclei have been preferred since in complex systems spectral resolution of the many different sites can be obtained and an armory of 2D techniques employed to give information about distances and bonding between neighboring atoms. The only NMR-active oxygen isotope, 17 O, has low natural abundance (0.037%) and spin (I ) 5 / 2 ). The resulting quadrupole interaction usually significantly broadens the signal. Even under magic-angle spinning (MAS), the line width for 17 O with large quadrupole interactions 2 means that signals from multiple sites are not readily resolved, so that site-specific information is masked, and many solid-state NMR techniques that depend on resolution and narrow lines cannot be applied. Consequently, solid-state 17 O NMR from complex biomolecules presents a significant challenge. Despite these difficulties, with the advance of high-field NMR spectrometers and methodologies, such as multi-quantum (MQ)-MAS 3 and double-rotation (DOR), 4 there has been a significant increase in solid-state 17 O NMR studies of inorganic 5-7 and organic/ biomaterials. [8][9][10][11][12][13][14] Here, by using 1 H-decoupled DOR, 17 O NMR signals are resolved from all eight similar, but distinct, oxygen sites in monosodium L-glutamate monohydrate (L-MSG), 16 which is used extensively as a food flavor enhancer. As shown in Figure 1a, the 17 O MAS spectrum (recorded at 14.1 T) shows a broad signal centered at ∼220 ppm with a line width of ∼8 kHz. The broad signal arises from the overlapping second-order quadrupole broadened lines with the presence of multiple sites indicated by the detailed features on the MAS envelope. However, with eight overlapping signals, spectral deconvolution to provide site-specific information is completely impracticable. In comparison, the 1 Hdecoupled 17 O DOR spectrum ( Figure 1b) exhibits major spectral improvement by successfully removing the second-order quadrupole broadening, producing seven sharp isotropic resonances with line widths less than 1 ppm, whose DOR isotropic positions (δ DOR ) are given in Table 1. The intensity for the resonance at 196 ppm is nearly twice that of the other six isotropic lines, suggesting that this resonance is from two oxygen sites. These sharp signals are ∼120 times narrower than those in the MAS spectrum. This is the first time that solid-state 17 O NMR has revealed eight distinct oxygen sites from a biomolecule. Previously, we have rep...

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

“…In particular, recent demonstrations on 17 O DOR spectroscopy have reported ultra-high spectral resolution with linewidths of <1 ppm). [15][16][17][18][19][20][21] It is worth noting that high-resolution 17 O NMR MQMAS and DOR have also been reported for many important inorganic materials. 5,[22][23][24] In comparison to inorganic materials, there are three major complications for 17 O solid-state NMR on bio/organic molecules: (1) the abundance of hydrogen in bio/organic molecules gives rise to significant dipolar broadening unless high power 1 H-decoupling is applied; (2) the quadrupole interaction is generally larger (7- 33 was used.…”

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

“…A number of studies have been reported utilizing DOR NMR for the differentiation of sodium, boron, aluminium and oxygen sites in minerals [9][10][11], materials [12][13][14][15][16] and molecular sieves, including, zeolites [17][18][19][20], sodalites [21][22][23] and aluminophosphates [24][25][26][27][28]. Recently, focus has turned to exploiting DOR for the study of oxygen sites in organic solids [29][30][31][32][33][34][35].…”

Determination of NMR interaction parameters from double rotation NMR

“…The majority of DOR NMR studies reported have been concerned with the differentiation of sodium, boron, aluminium and oxygen sites in minerals [18][19][20], materials [21][22][23][24][25] and molecular sieves, including, zeolites [26][27][28][29], sodalites [30][31][32] and aluminophosphates [33][34][35][36][37]. More recently, focus has turned to exploiting DOR for the study of oxygen sites in organic solids [17,[38][39][40][41].…”