Journal
CHEMPHYSCHEM
Volume 18, Issue 19, Pages 2697-2703Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cphc.201700572
Keywords
NMR methods; NMR pulse sequences; phage proteins; protein assemblies; protein quality control machinery
Funding
- European Research Council [ERC-StG-311318-ProtDyn2Function, ERC-StG-2010-260887]
- INSTRUCT (Innovative EM/NMR approach for the characterization of the drug target ClpP APPID: 301)
- FRISBI [ANR-10-INSB-05-02]
- GRAL [ANR-10-LABX-49-01]
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Solid-state NMR spectroscopy 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 the use of two sets of experiments is shown to expand the tool kit of H-1-detected assignment approaches, which correlate a given amide pair either to the two adjacent CO-CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide H-1 of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross-polarization for heteronuclear transfers. The utility of these experiments is exemplified with application to assemblies of deuterated, fully amide-protonated proteins from approximately 20 to 60kDa monomer, at magic-angle spinning (MAS) frequencies from approximately 40 to 55kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. The resonance assignment of a domain within the 50.4kDa bacteriophageT5 tube protein pb6 is reported, and this is compared to NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly.
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