Conquering poor resolution in fast magic angle spinning solid-state NMR spectra of noncrystallisable protein assemblies.
This study aimed to develop a technique to tackle inhomogeneous broadening, a major cause in the loss of site-specific information in solid-state NMR, using a technique termed saturation exchange spectroscopy (SEXY).
Figure 1. Saturation exchange spectroscopy鈥檚 application in inhomogeneous broadening. NMR requires the absorbance of RF radiation to cause 鈥榝lipping鈥� of nuclear spins between two spin states (in the case of 13C) with a population imbalance. Frequency-selective RF pulses, of sufficiently high energy, irradiate single peaks within a broadened region resulting in equalisation of populations within both spin states. Thus, the NMR signal sharply decreases (saturates). Saturation transfer subsequently occurs via spin diffusion, a process driven by dipolar coupling (through space interactions), resulting in the loss of signal of coupled peaks. Spin diffusion arises among nuclear spins of the same kind (like-spins) within a few to several Angstroms in space.11 Therefore, previously unattainable structural information, from broadened peaks, is now accessible. Spin diffusion occurs due to the presence of Ii+Ij- and Ii-Ij+ terms within the dipolar spin Hamiltonian between two like-spins.11,12 These terms cause mutual flipping of two nearby spins resulting in a transfer of polarisation from the first spin to the second. This mechanism causes propagation of spin polarisation until it is lost by longitudinal relaxation (T1) processes.
Proof-of-concept was obtained using N-formyl-Met-Leu-Phe-OH (MLF), a simple tripeptide, whereby sites on the amino acid residues were selectively saturated and saturation transfer was observed.
Figure 4. Two-dimensional 13C SEXY spectra of fMLF. Each row in the indirect dimension of the spectra consists of a saturation pulse at a specific frequency. (b) An easy to interpret difference spectrum was obtained by subtracting each row of (a) the original data by a 1D unsaturated spectrum. (c) The aliphatic region was of most interest as extensive saturation transfer is visible. In total, 17 saturation pulses were applied, two of which were off-resonance, at the following frequencies (ppm): 188.9 (1), 174.8, 173.2, 172.0, 135.6, 130.0, 127.8, 56.8, 54.3, 52.1, 40.6, 37.6, 28.8, 24.8, 19.7, 14.1 and -9.9 (17).
Two-dimensional 13C SEXY spectra obtained at fast and slow magic angle spinning frequencies were comparable to 2D 13C-13C Proton-driven spin diffusion (PDSD) spectra suggesting that SEXY was well optimised and could also be used for structural assignment.
Figure 8. Two-dimensional 13C spectra obtained using (a) PDSD and (b) SEXY. A mixing/ saturation time of 0.300 s was utilised. The total analysis time for the PDSD experiment was ~3.5 hours and ~1.5 hours for the SEXY experiment.
Directing future studies on the analysis of complex inhomogenous systems would determine if SEXY is a suitable approach for tackling inhomogeneous broadening, one of ssNMR's greatest issues.