Proton assisted recoupling and protein structure determination

Abstract

We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between (13)C or (15)N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between (1)H-(13)C (or (1)H-(15)N) couplings to mediate zero quantum (13)C-(13)C (or (15)N-(15)N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C(1) (+/-)C(2) (-/+)H(Z) for (13)C-(13)C recoupling experiments (or N(1) (+/-)N(2) (-/+)H(Z) for (15)N-(15)N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with (13)C-(13)C spectra of uniformly (13)C, (15)N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2 x 10.4 kDa). The spectra, which were acquired at high MAS frequencies (omega(r)2pi>20 kHz) and magnetic fields (750-900 MHz (1)H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A) (13)C-(13)C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein.

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.…”

Heteronuclear proton assisted recoupling

“…This protocol has yielded several MAS NMR structures with a precision ≤1 Å. 3,[12][13][14][15] Most heteronuclear experiments require 15 N-13 C dipolar couplings to be reintroduced with a train of π pulses at the 13 C and/or 15 N frequencies while simultaneously decoupling the 1 H spins from the 15 N-13 C spin dynamics. This is the basic approach employed in the development of the majority of heteronuclear recoupling sequences including rotational echo double resonance (REDOR), 7 transferred echo double resonance (TEDOR), 8 frequency selective (FS)-REDOR, 9 z-filtered (ZF-) and band-selective (BASE-)TEDOR, 9 and frequency selective (FS-)TEDOR 16 which enable accurate N-C distance measurements, and have been especially important in the determination of high resolution 3D structures.…”

“…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.…”