Proton Assisted Insensitive Nuclei Cross Polarization

Abstract

This communication presents a solid-state NMR 15 N-13 C polarization transfer scheme applicable at high B 0 and high MAS frequencies, requiring moderate r.f. powers (~50 kHz 13 C/ 15 N) and mixing time (1-6 ms). The sequence, PAIN-CP, involves the abundant nearby protons in the heteronuclear recoupling dynamics, and provides a new tool for obtaining long distance 15 N-13 C contacts. It should be of major interest for biomolecular structural studies.Polarization transfer 1-15 between different nuclear species mediated by dipolar couplings is used extensively in magic angle spinning (MAS) 16 experiments to perform chemical shift assignments, and to measure distances 5-7, 11, 17-21 and torsion angles. 22-25 Heteronuclear dipolar recoupling sequences can be classified into two categories depending on their behavior with respect to dipolar truncation. The first group includes the double CP sequence (DCP26) and its variants (SPICP27, RFDRCP 4 , iDCP 9 ) which lead to non-commuting terms in the effective Hamiltonian, and thus are mainly used to perform one-bond transfers (NCO, NCA) sometimes followed by a homonuclear 13 C-13 C recoupling period for obtaining 15 N-13 C multiple-bond contacts. 28, 29 The second group of sequences (REDOR5, TEDOR 1 / REPT 19 , GATE 17 ) yields a longitudinal effective Hamiltonian and enables measurement of long distances (< 4 Å). 20, 21High magnetic fields (>600 MHz) are an important experimental component for improving sensitivity and resolution in biomolecular MAS experiments involving 15 N-13 C magnetization transfer, provided that experiments can be performed at high MAS frequencies (>15 kHz) to compensate for increases in chemical shift anisotropies (CSA). Unfortunately, the application of the sequences mentioned above becomes problematic in this regime as the applied high r.f. powers lead to increased sample heating, jeopardize the integrity of the probe, but often do not provide sufficient 1 H decoupling.Here we present an efficient 15 N-13 C heteronuclear recoupling technique that involves nearby protons in the transfer and is applicable at high MAS frequency (ω r /2π>20 kHz). This new scheme, Proton Assisted Insensitive Nuclei Cross Polarization (PAIN-CP), reduces dipolar truncation and therefore is particularly well suited for obtaining long distance contacts. PAIN-CP demonstrates that the involvement of protons in the polarization transfer between low-γ nuclei does not have to be deleterious in nature; on the contrary 1 H's can be used to enhance the rate and efficiency of the transfer. The PAIN-CP experiment utilizes a mechanism which we refer to as Third Spin Assisted Recoupling (TSAR). Its extension to the homonuclear case is straightforward and will be discussed elsewhere. Note that the use of 1 H irradiation has been reported previously for 13 C-13 C recoupling experiments, 30-32 but that the underlying mechanism is different. Even though PAIN-CP and DCP 26 have similar pulse sequences (see Figure 1 and Supporting Information Section 7-8 (S.I.-7, 8)), they rely on v...

Dipolar truncation in magic-angle spinning NMR recoupling experiments

“…2 69 We restricted our investigation to zeroth-order dipolar recoupling sequences that produce a zeroth-order recoupled dipolar Hamiltonian, which are the ones typically used to obtain accurate internuclear distance measurements. We specifically avoided the treatment of proton-driven spin diffusion, [70][71][72] DARR/RAD, [73][74][75] homonuclear TSAR 22,23 techniques and MIRROR 25 recoupling, which perform polarization transfer via higher-order mechanisms that are more tolerant to dipolar truncation effects in multiple-spin systems, as shown previously, 22,51 but typically yield ambiguous distance constraints due to the complexity of the mechanisms.…”

“…In this publication we focus primarily on zeroth-order sequences. Second-order or higher-order recoupling sequences [22][23][24][25] have different properties because they contain cross terms between two interactions and the effective coupling constants depend on both interactions.…”

13C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies

“…In this regard, we found that the TSAR mechanism 35 constitutes an excellent choice to transfer the polarization from 15 N (residue i) to neighboring 13 C atoms in a bidirectional way (i.e., to residues i À 1 and i þ 1). 2D PAIN-CP spectra (Figure 4a) can be acquired in a relatively short experimental time (1À2 days), leading to highly resolved 15 NÀ 13 C spectra.…”

Heteronuclear proton assisted recoupling

“…18,19 Recently, a number of different methods were demonstrated as useful for providing long-range distance restraints for protein structure determination. 14,[20][21][22] Among them there are two promising approaches: proton assisted recoupling (PAR) and proton assisted insensitive nuclei cross polarization (PAIN-CP), which are based on a more general third spin assisted recoupling (TSAR) mechanism. 14,22 Notably, these new recoupling methods have been used to solve the largest solid-state nuclear magnetic resonance (SSNMR) protein structure reported to date (i.e., .…”

“…14,[20][21][22] Among them there are two promising approaches: proton assisted recoupling (PAR) and proton assisted insensitive nuclei cross polarization (PAIN-CP), which are based on a more general third spin assisted recoupling (TSAR) mechanism. 14,22 Notably, these new recoupling methods have been used to solve the largest solid-state nuclear magnetic resonance (SSNMR) protein structure reported to date (i.e., . 12 These techniques have also been applied to the challenging case of 15 N-15 N correlation spectroscopy and also demonstrated as one of the few pulse sequences able to provide long distance restraints in uniformly 13 C and 15 N labeled proteins in the ω r /2π > 50 kHz spinning frequency regime.…”