Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range¹³C-¹³C and¹⁵N-¹³C Distance Restraints

Abstract

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 1 H-13 C and 1 H-15 N dipolar couplings to achieve 13 C-13 C and 13 C-15 N 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 PERSPIRATION CP (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 Å for 13 C-13 C and 15 N-13 C spin pairs, respectively. We also apply these methods to the amyloid fibrils formed by the peptide hormone glucagon, and show that longrange correlation peaks are readily observed to constrain intermolecular packing in this cross-b fibril.We provide an analytical model for the PULSAR and PERSPIRATION CP 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.Third-Spin-Assisted Recoupling (TSAR) experiments are a class of techniques that transfer polarization between low-g nuclei A and B 27 . This transfer occurs via cross terms in the second-order average Hamiltonian between the H-A and H-B dipolar couplings, which are generally much stronger than the direct A-B dipolar coupling. In continuous-wave TSAR (CW-TSAR) experiments, the homonuclear variant is known as Proton-Asisted Recoupling (PAR) 28 whereas the heteronuclear variant is called Proton-Asissted Insensitive Nuclei Cross Polarization ( PAIN CP) [29][30] . The CW irradiation in these experiments requires carefully optimized rf field strengths on both the 1 H channel and the heteronuclear channels. This is usually achieved with an extensive multidimensional search of the rf field strengths, without which the polarization transfer efficiency can easily fall below the theoretical maximum 31 . Moreover, mixing times of 10-30 ms are usually required to observe long-range 13 C-13 C and 15 N-13 C correlation peaks. The extended high-power CW irradiation on two to three channels can damage the NMR probe and heat-sensitive biological samples. These two practical drawbacks have limited the use of the PAR and PAIN CP experiments, despite their theoretical ability for long-range spin polarization transfer, compared to the simpler spin diffusion experiments.Here we introduce pulsed TSAR (P-TSAR...