DOI: 10.1002/cbic.201000124 Protocols for the Sequential Solid-State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET-s(1-227) Anne Schuetz, [a] Christian Wasmer, [a] Birgit Habenstein, [b] RenØ Verel, [a] Jason Greenwald, [a] Roland Riek, [a] Anja Bçckmann,* [b] and Beat H. Meier* [a] Introduction Solid-state NMR spectroscopy has over the last few years de- veloped to a point at which a breakthrough in the field of structure determination for insoluble peptides and proteins is forthcoming. Indeed, the first atomic-resolution 3D structures determined from solid-state NMR spectroscopic data have been presented recently. [1–8] So far, these proteins have all been smaller than 100 amino acid residues, and the progress towards the structure determination of larger membrane and fibrillar proteins will require the adaptation and extension of the current protocols. The sequential resonance assignment, that is, the correlation of the NMR frequencies with the nuclear spins of each amino acid in the primary sequence, is the basis of all further NMR spectroscopy studies, such as site-specific dynamics, interaction measurements and 3D structure determi- nation. A growing number of sequential resonance assignments of solid-state spectra from uniformly labeled proteins are avail- able today. [9–17] Most of these assignments were achieved from 2D spectroscopy, but the resolution in such spectra severely limits the size of the proteins that can be addressed. Conse- quently, the few available assignments of larger proteins (> 200 amino acids) were rendered feasible by employing spe- cific isotope-labeling schemes, for example, reverse labeling of only a subset of spins, [18–21] or checkerboard labeling schemes, [22] in combination with three-dimensional spectroscopy. Herein we describe an assignment protocol based on three- dimensional NMR spectra that can be applied to larger, uni- formly [ 13 C, 15 N]-labeled proteins. It could also be combined with specific labeling schemes and extended to even larger proteins. The protocol we present is based on a number of previously developed pulse schemes [23–29] that were chosen to provide optimum efficiencies for the three or more polariza- tion-transfer steps involved. In addition, our approach takes advantage of recent advances in spectrometer technology and sample preparation to achieve the necessary resolution and sensitivity in the experiments described. High static magnetic fields, optimized probe heads, and advances in sample prepa- ration, including optimized filling of the protein into the rotor, [30] have brought better resolution and a substantial in- crease of the signal-to-noise ratio. These advances currently allow for the recording of 3D spectra that include up to four transfer steps between heteronuclei within 3–5 days by using a sample of less than 25 mg of a 25 kDa protein. The HET-s prion from the fungus Podospora anserina is in- volved in a genetically controlled cell-death reaction (hetero- karyon incompatibility) and consists of two domains, a prion domain and a globular domain (1–227). [31] In the following, we study the globular domain, HET-s(1–227), of which the crystal structure has recently been solved. [32] The protein consists of eight a helices and three short 3 10 helices, accounting for the vast majority of residues. It has very little b-sheet content with only two strands of three residues each. The rather uniform secondary structure content makes it a challenging system for [a] A. Schuetz, C. Wasmer, Dr. R. Verel, Dr. J. Greenwald, Prof. R. Riek, Prof. B. H. Meier Physical Chemistry, ETH Zürich 8093 Zürich (Switzerland) E-mail : beme@ethz.ch [b] B. Habenstein, Dr. A. Bçckmann Institut de Biologie et Chimie des ProtØines, UMR 5086 CNRS/UniversitØ de Lyon 1 7 passage du Vercors, 69367 Lyon (France) E-mail : anja.bockmann@ibcp.fr Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000124. The sequence-specific resonance assignment of a protein forms the basis for studies of molecular structure and dynam- ics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential 13 C and 15 N reso- nance assignment of uniformly [ 15 N, 13 C]-labeled proteins, based on a suite of complementary three-dimensional solid-state NMR spectroscopy experiments. It is directed towards the ap- plication to proteins with more than about 100 amino acid res- idues. The assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitro- gen and carbon spins, including the well-dispersed C b resonan- ces. Supplementary spectra that correlate further side-chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demon- strate the application of this assignment protocol for a crystal- line preparation of the N-terminal globular domain of the HET- s prion, a 227-residue protein. ChemBioChem 2010, 11, 1543 – 1551  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1543