Out-and-back 13C–13C scalar transfers in protein resonance assignment by proton-detected solid-state NMR under ultra-fast MAS (original) (raw)
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Journal of the American Chemical Society, 2014
Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5−30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.
2013
Resonance assignment is the first step in NMR structure determination. For magic angle spinning NMR, this is typically achieved with a set of heteronuclear correlation experiments (NCaCX, NCOCX, CONCa) that utilize SPECIFIC-CP 15 N-13 C transfers. However, the SPECIFIC-CP transfer efficiency is often compromised by molecular dynamics and probe performance. Here we show that one-bond ZF-TEDOR 15 N-13 C transfers provide simultaneous NCO and NCa transfers with at least as much sensitivity as SPECIFIC-CP for some non-crystalline samples. Furthermore, a 3D TEDOR-CC experiment provides heteronuclear sidechains correlations and robustness with respect to proton decoupling and radiofrequency power instabilities. We demonstrate transfer efficiencies and connectivities by application of 3D ZF-TEDOR-DARR to a model microcrystalline protein, GB1, and a less ideal system, GvpA in intact gas vesicles.
Journal of Biomolecular NMR, 2014
Heteronucleus-detected dipolar based correlation spectroscopy is established for assignments of 1 H, 13 C, and 15 N resonances and structural analysis in fully protonated proteins. We demonstrate that 13 C detected 3D experiments are highly efficient and permit assignments of the majority of backbone resonances, as shown in an 89-residue dynein light chain 8, LC8 protein. With these experiments, we have resolved many ambiguities that were persistent in our previous studies using moderate MAS frequencies and lacking the 1 H dimension. The availability of 1 H isotropic chemical shifts measured with the heteronucleus-detected fast-MAS experiments presented here is essential for the accurate determination of the 1 H CSA tensors, which provide very useful structural probe. Finally, our results indicate that 13 C detection in fast-MAS HETCOR experiments may be advantageous compared with 1 H detection as it yields datasets of significantly higher resolution in the 13 C dimension than the 1 H detected HETCOR versions.
A Resonance Assignment Method for Oriented-Sample Solid-State NMR of Proteins
Journal of the American Chemical Society, 2010
Oriented sample (OS) solid-state NMR is capable of determining the three-dimensional structures of proteins in their native functional environments when they are immobilized and aligned in supramolecular assemblies 1 , such as virus particles or membranes. Examples include gramicidin 2 , AchR M2 domain 3 , M2 domain of the influenza A virus 4 , fd 5 and Pf1 6 phage coat proteins, phospholamban 7 , Vpu (from HIV-1) 8 , and MerF 9 .
Journal of The American Chemical Society, 2007
Remarkable progress in solid-state NMR has enabled complete structure determination of uniformly labeled proteins in the size range of 5-10 kDa. Expanding these applications to larger or masslimited systems requires further improvements in spectral sensitivity, for which inverse detection of 13 C and 15 N signals with 1 H is one promising approach. Proton detection has previously been demonstrated to offer sensitivity benefits in the limit of sparse protonation or with ∼30 kHz magic-angle spinning (MAS).
Chem. Sci., 2011
We introduce a new experiment, which makes use of Spin State Selective manipulations to perform sensitive and resolved through-bond correlations in organic and biological solids at high-fields and under ultra-fast MAS. The scheme is the shortest and most sensitive through-bond correlation method introduced so far in solids, yields resolved fingerprints of uniformly 13 C-labeled biomolecules, and constitutes a tool to highlight slight static structural disorder around crystallographically equivalent molecules in microcrystalline samples.