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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 degrees C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, H-1-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 traditional C-13-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 by 1H-detected ssNMR. It requires a perdeuterated, uniformly 13C-and N-15-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.