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

We introduce a class of pulsed third-spin-assisted recoupling (P-TSAR) magic-angle-spinning (MAS) solid-state NMR techniques that achieve efficient polarization transfer over long distances to provide important restraints for structure determination. These experiments operate with the same principle as continuous-wave (CW) TSAR experiments, by utilizing second-order cross terms between strong 1H-13C and 1H-15N dipolar couplings to achieve 13C-13C and 13C-15N polarization transfer. However, in contrast to the CW-TSAR experiments, these pulsed P-TSAR experiments require much less radiofrequency (rf) energy and allow a much simpler routine for optimizing the rf field strength. We call the techniques PULSAR (PULSed proton Asissted Recoupling) for homonuclear spin pairs and PERSPIRATIONCP (Proton-Enhanced Rotor-echo Short Pulse IRradiATION Cross-Polarization) for heteronuclear spin pairs. We demonstrate these techniques on the model protein GB1, and found cross peaks for distances as long as 10 and 8 [A] for 13C-13C and 15N-13C spin pairs, respectively. We also 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-{beta} 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.