Using a set of six ¹H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide ²H/¹H exchange, high magnetic fields, and high-spinning frequencies (ω_r/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary ¹³C/¹⁵N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

Significance: Protein structure determination is key to the detailed description of many biological processes. The critical factor that would allow general application of magic-angle spinning (MAS) solid-state NMR to this end is improvement in sensitivity and resolution for as many nuclear spins as possible. This is achieved here with detection of resolved ¹ H resonances in protonated proteins by increasing MAS rates to frequencies of 100 kHz and above. For large proteins and assemblies, ultrafast spinning narrows spectral resonances better than Brownian motion on which solution NMR relies, removing a fundamental barrier to the NMR study of large systems. This is exploited here to determine the de novo structure of a 28-kDa protein dimer in a 2.5-MDa viral capsid assembly.

Expanding the bacterial toxin-antitoxin system classes to a fifth class, GhoST was found to be involved in maintenance of persister cells, dormant cells that are tolerant of antibiotics. GhoS is the antitoxin, an endoribonuclease that cleaves the toxin mRNA ghoT, whose gene product is a membrane-lytic protein. Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

The NMR structures of the recombinant cellular form of the prion proteins (PrP ^C ) of the cat ( Felis catus ), dog ( Canis familiaris ), and pig ( Sus scrofa ), and of two polymorphic forms of the prion protein from sheep ( Ovis aries ) are presented. In all of these species, PrP ^C consists of an N-terminal flexibly extended tail with ≈100 amino acid residues and a C-terminal globular domain of ≈100 residues with three α-helices and a short antiparallel β-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrP ^C structures, there are local differences between the globular domains of the different species. Because the five newly determined PrP ^C structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrP ^C three-dimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies.