Prions are believed to be infectious self-propagating polymers of otherwise soluble host-encoded proteins 1,2 . This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast 3-5 , Podospora anserina 6 , and mammals 7 can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a C-terminal prion domain comprising residues 218-289. Amyloid fibrils of are necessary and sufficient for the induction and propagation of prion infectivity 6 . Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid state NMR to determine the sequence specific positions of secondary structure elements of amyloid fibrils of . This revealed four β-strands constituted by two pseudo repeat sequences, each forming a β-strandturn-β-strand motif. We show that this conformation is the functional and infectious entity of the HET-s prion by using a structure-based mutagenesis approach. These results correlate for the first time distinct structural elements with prion infectivity.The prion form of the protein HET-s is involved in a programmed cell death phenomenon termed heterokaryon incompatibility 8,9 . This reaction occurs in filamentous fungi when cells of incompatible genotype fuse and form a mixed cell. In P. anserina, two incompatible genotypes, called het-s and het-S, encode for the proteins HET-s and HET-S. They are both 289 amino acids long and differ in only 13 residues 10 . However, only HET-s can form a prion 11 : P. anserina cells expressing the HET-s protein exist either in a prion state called [Hets] Correspondence and requests for materials should be addressed to R.R. (e-mail: riek@salk.edu).. $ these authors contributed equally to this work.Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). (Fig. 1a) contained one assigned cross-peak for each backbone amide of with the exception of 289, enabling a residuespecific determination of the hydrogen exchange rates. Upon exchange in D 2 O buffer for 6 weeks the intensity of about 45% of the resonances was significantly reduced or absent from the spectrum (Fig. 1b). This suggested that the corresponding amides have undergone exchange with solvent deuterons, which are not visible in this experiment. NIH Public AccessThe hydrogen exchange was followed over a total period of 3 months. All residues displayed a mono-exponential decay (Fig. S1) indicating that the structure of the fibrils was well defined and homogeneous. The summarized hydrogen exchange data (Fig. 1e) show that due to exchange rates faster than 5·h -1 , the 8 N-terminal residues, the 5 C-terminal residues and residues 247-261 are only weakly or not protected and may therefore be conformationally disordered. Four segments were observed that d...

Magic-angle spinning solid-state NMR spectroscopy is an important technique to study molecular structure, dynamics and interactions, and is rapidly gaining importance in biomolecular sciences. Here we provide an overview of experimental approaches to study molecular dynamics by MAS solid-state NMR, with an emphasis on the underlying theoretical concepts and differences of MAS solid-state NMR compared to solution-state NMR. The theoretical foundations of nuclear spin relaxation are revisited, focusing on the particularities of spin relaxation in solid samples under magic-angle spinning. We discuss the range of validity of Redfield theory, as well as the inherent multi-exponential behavior of relaxation in solids. Experimental challenges for measuring relaxation parameters in MAS solid-state NMR and a few recently proposed relaxation approaches are discussed, which provide information about time scales and amplitudes of motions ranging from picoseconds to milliseconds. We also discuss the theoretical basis and experimental measurements of anisotropic interactions (chemical-shift anisotropies, dipolar and quadrupolar couplings), which give direct information about the amplitude of motions. The potential of combining relaxation data with such measurements of dynamically-averaged anisotropic interactions is discussed. Although the focus of this review is on the theoretical foundations of dynamics studies rather than their application, we close by discussing a small number of recent dynamics studies, where the dynamic properties of proteins in crystals are compared to those in solution.

Solid-state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane-associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10-20 mg each. Here we show that a new NMR probe, pushing magic-angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well-defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 μg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in-cell or cell-free expression.

Characterization of protein dynamics by solid-state NMR spectroscopy requires robust and accurate measurement protocols, which are not yet fully developed. In this study, we investigate the backbone dynamics of microcrystalline ubiquitin using different approaches. A rotational-echo double-resonance type (REDOR-type) methodology allows one to accurately measure (1)H-(15)N order parameters in highly deuterated samples. We show that the systematic errors in the REDOR experiment are as low as 1% or even less, giving access to accurate data for the amplitudes of backbone mobility. Combining such dipolar-coupling-derived order parameters with autocorrelated and cross-correlated (15)N relaxation rates, we are able to quantitate amplitudes and correlation times of backbone dynamics on picosecond and nanosecond time scales in a residue-resolved manner. While the mobility on picosecond time scales appears to have rather uniform amplitude throughout the protein, we unambiguously identify and quantitate nanosecond mobility with order parameters S(2) as low as 0.8 in some regions of the protein, where nanosecond dynamics has also been revealed in solution state. The methodology used here, a combination of accurate dipolar-coupling measurements and different relaxation parameters, yields details about dynamics on different time scales and can be applied to solid protein samples such as amyloid fibrils or membrane proteins.

Magic-angle spinning (MAS) is a technique that is a prerequisite for high-resolution solid-state NMR spectroscopy of proteins and other biomolecules. Recently, the 100 kHz limit for the rotation frequency has been broken, arguably making MAS rotors the man-made objects with the highest rotation frequency. This development is expected to have a significant impact on biomolecular NMR as it facilitates proton detection, which allows to partially compensate the loss in overall sensitivity associated with the small sample amounts that fit into MAS rotors with less than 1 mm outer diameter. Under these conditions, the mass-normalized sensitivity of a small rotor becomes much higher than that of larger-volume rotor.2