Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range ¹³C–¹³C and ¹⁵N–¹³C Distance Restraints

We present a class of pulsed third-spin-assisted recoupling (P-TSAR) magic-angle-spinning solid-state NMR techniques that achieve efficient polarization transfer over long distances to provide important restraints for structure determination. These experiments utilize second-order cross terms between strong ¹H–¹³C and ¹H–¹⁵N dipolar couplings to achieve ¹³C–¹³C and ¹⁵N–¹³C polarization transfer, similar to the principle of continuous-wave (CW) TSAR experiments. However, in contrast to the CW-TSAR experiments, these P-TSAR experiments require much less radiofrequency (rf) energy and allow a much simpler routine for optimizing the rf field strength. We call the technique PULSAR (pulsed proton-assisted recoupling) for homonuclear spin pairs. For heteronuclear spin pairs, we improve the recently introduced ^PERSPIRATIONCP (proton-enhanced rotor-echo short pulse irradiation cross-polarization) experiment by shifting the pulse positions and removing the z-filters, which significantly broaden the bandwidth and increase the efficiency of polarization transfer. We demonstrate the PULSAR and ^PERSPIRATIONCP techniques on the model protein GB1 and found cross peaks for distances as long as 10 and 8 Å for ¹³C–¹³C and ¹⁵N–¹³C spin pairs, respectively. We then apply these methods to the amyloid fibrils formed by the peptide hormone glucagon and show that long-range correlation peaks are readily observed to constrain intermolecular packing in this cross-β fibril. We provide an analytical model for the PULSAR and ^PERSPIRATIONCP experiments to explain the measured and simulated chemical shift dependence and pulse flip angle dependence of polarization transfer. These two techniques are useful for measuring long-range distance restraints to determine the three-dimensional structures of proteins and other biological macromolecules.