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Solid‐state NMR H‐N‐(C)‐H and H‐N‐C‐C 3D/4D correlation experiments for resonance assignment of large proteins
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Edité par CCSD ; Wiley-VCH Verlag -
International audience. Solid-state NMR can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid-state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here we show the use of two sets of experiments that expand the tool kit of 1H-detected assignment approaches, and which correlate a given amide pair either to the two adjacent CO-CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to up to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross-polarization for heteronuclear transfers. We exemplify the usefulness of these experiments with applications to assemblies of deuterated, fully amide-protonated proteins from ca. 20 to 60 kDa monomer, at MAS frequencies from ca. 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. We report the resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6, and compare these to solution-state NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly. Solid‐state NMR H‐N‐(C)‐H and H‐N‐C‐C 3D/4D correlation experiments for resonance assignment of large proteins. Available from: https://www.researchgate.net/publication/319020315_Solid-state_NMR_H-N-C-H_and_H-N-C-C_3D4D_correlation_experiments_for_resonance_assignment_of_large_proteins.
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