title: Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning.
abstract: Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 °C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years,1H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue. Such methods are, however, substantially more demanding to the spectroscopist, as compared with traditional13C-detected approaches. As a guide, this protocol describes a procedure for the chemical shift assignment of the backbone atoms of proteins in the solid state by1H-detected ssNMR. It requires a perdeuterated, uniformly13C- and15N-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues; taken together, these correlations allow for complete chemical shift assignment via a 'backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/or dynamics by ssNMR. Depending on the spectral quality and complexity of the protein, data acquisition and analysis are possible within 2 months.
authors.affiliation: Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.
authors.forename: Pascal
authors.initials: P
authors.lastname: Fricke
authors.affiliation: Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.
authors.forename: Veniamin
authors.initials: V
authors.lastname: Chevelkov
authors.affiliation: Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Zinke
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Giller
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.|Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany.
authors.forename: Adam
authors.initials: A
authors.lastname: Lange
mesh_terms: D008051:Lipid Bilayers / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011506:Proteins / Q000737:chemistry; D011522:Protons; D013696:Temperature; D013997:Time Factors
chemical_list: D008051:Lipid Bilayers; D011506:Proteins; D011522:Protons