Heteronuclear proton assisted recoupling

Abstract

We describe a theoretical framework for understanding the heteronuclear version of the third spin assisted recoupling polarization transfer mechanism and demonstrate its potential for detecting longdistance intramolecular and intermolecular 15 N-13 C contacts in biomolecular systems. The pulse sequence, proton assisted insensitive nuclei cross polarization (PAIN-CP) relies on a cross term between 1 H-15 N and 1 H-13 C dipolar couplings to mediate zero-and/or double-quantum 15 N-13 C recoupling. In particular, using average Hamiltonian theory we derive effective Hamiltonians for PAIN-CP and show that the transfer is mediated by trilinear terms of the form N ± C ∓ H z (ZQ) or N ± C ± H z (DQ) depending on the rf field strengths employed. We use analytical and numerical simulations to explain the structure of the PAIN-CP optimization maps and to delineate the appropriate matching conditions. We also detail the dependence of the PAIN-CP polarization transfer with respect to local molecular geometry and explain the observed reduction in dipolar truncation. In addition, we demonstrate the utility of PAIN-CP in structural studies with 15 N-13 C spectra of two uniformly 13 C, 15 N labeled model microcrystalline proteins-GB1, a 56 amino acid peptide, and Crh, a 85 amino acid domain swapped dimer (MW = 2 × 10.4 kDa). The spectra acquired at high magic angle spinning frequencies (ω r /2π > 20 kHz) and magnetic fields (ω 0H /2π = 700-900 MHz) using moderate rf fields, yield multiple long-distance intramonomer and intermonomer 15 N-13 C contacts. We use these distance restraints, in combination with the available x-ray structure as a homology model, to perform a calculation of the monomer subunit of the Crh protein.

Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra

“…Additionally, although we assessed the performance of MEIOSIS for specific-CP 15 N-13 C transfer, we anticipate that it could also be extended to other 15 N-13 C polarization transfer schemes such as symmetry based transfer sequences, 25 PAIN-CP (proton assisted insensitive nuclei cross polarization), as well as optimal control based NC transfer. 26,27 As recently demonstrated for the DU-MAS methods, 28 we anticipate that the variants of MEIOSIS pulse schemes will be implemented for the structural analysis of U-2 H, 13 C, 15 N proteins with 1 H and 13 C dual-receiver acquisition.…”

Mechanism of dilute-spin-exchange in solid-state NMR

“…Here we demonstrate that an alternative mechanism is operative, namely polarization transfer through 1 H-1 H coupling. Notably, this mechanism does not require that each 1 H nucleus be coupled to both 15 N nuclei. Moreover, it more closely reflects the distribution of nuclei in polypeptides where 15 N nuclei are usually located on different residues; only rarely does a 1 H nucleus have sizable dipolar couplings to two or more 15 N nuclei.…”

“…Notably, this mechanism does not require that each 1 H nucleus be coupled to both 15 N nuclei. Moreover, it more closely reflects the distribution of nuclei in polypeptides where 15 N nuclei are usually located on different residues; only rarely does a 1 H nucleus have sizable dipolar couplings to two or more 15 N nuclei. By comparing the effects of on-resonance versus "magic-angle" off-resonance 1 H spin-lock irradiation 37 it is possible to separate the two transfer mechanisms for dilutespin-exchange.…”

“…Spin-exchange between 15 N nuclei is frequently utilized in protein NMR studies for resonance assignment and structure determination because of the importance of the amide sites located in the polypeptide backbone. TSAR was adapted for stationary, aligned samples in oriented sample (OS) solidstate NMR as the mismatched Hartmann-Hahn (MMHH) condition.…”

“…TSAR was adapted for stationary, aligned samples in oriented sample (OS) solidstate NMR as the mismatched Hartmann-Hahn (MMHH) condition. 25 TSAR and several other second-order recoupling experiments, [32][33][34] including proton driven spin diffusion (PDSD), 35,36 rely on the presence of nearby 1 H nuclei to transfer magnetization between 15 N sites. The 1 H nucleus participation in the magnetization transfer is proposed to be through simultaneous coupling to both 15 N nuclei.…”