Heteronuclear proton assisted recoupling

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 long-distance 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.