Solid‐state NMR spectroscopy can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid‐state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here the use of two sets of experiments is shown to expand the tool kit of 1H‐detected assignment approaches, which correlate a given amide pair either to the two adjacent CO–CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross‐polarization for heteronuclear transfers. The utility of these experiments is exemplified with application to assemblies of deuterated, fully amide‐protonated proteins from approximately 20 to 60 kDa monomer, at magic‐angle spinning (MAS) frequencies from approximately 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. The resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6 is reported, and this is compared to NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly.

The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and prevents premature release of the RNA from the enzyme. Using methyl TROSY NMR techniques, we establish that the 3′ end of the substrate remains highly flexible inside the lumen. As a result, the RNA jumps between the three active sites that all equally participate in substrate degradation. The RNA jumping rate is, however, much faster than the cleavage rate, indicating that not all active site:substrate encounters result in catalysis. Enzymatic turnover therefore benefits from the confinement of the active sites and substrate in the lumen, which ensures that the RNA is at all times bound to one of the active sites. The evolution of the exosome into a hexameric complex and the optimization of its catalytic efficiency were thus likely co-occurring events.

Big and unexpectedly flexible: The 173 kDa exosome core is unexpectedly dynamic in solution. The kinetic and thermodynamic properties connected with the identified exchange process were quantified. Both cap protein and RNA substrate binding significantly alter the identified motions, suggesting that these interactions occur through conformational selection.

The exosome is a large molecular machine that is involved in RNA degradation and processing. Here, we address how the trimeric Rrp4 cap enhances the activity of the archaeal enzyme complex. Using methyl TROSY NMR methods we identified a 50 Å long RNA binding path on each Rrp4 protomer. We show that the Rrp4 cap can thus recruit three substrates simultaneously, one of which is degraded in the core while two others are positioned for subsequent degradation rounds. The local interaction energy between the substrate and the Rrp4-exosome increases from the periphery of the complex towards the active sites. Importantly, the intrinsic interaction strength between the cap and the substrate is weakened as soon as substrates enter the catalytic barrel, which provides a means to reduce friction during substrate movements towards the active sites. Our data thus reveal a sophisticated exosome–substrate interaction mechanism that enables efficient RNA degradation.

Groß und unerwartet flexibel: Der 173‐kDa‐Exosomkernkomplex erweist sich in Lösung als unerwartet dynamisch. Die kinetischen und thermodynamischen Daten des identifizierten Austauschprozesses wuerden bestimmt. Die Bindung eines Cap‐Proteins wie auch die eines RNA‐Substrats verändern die Bewegungen deutlich, was dafür spricht, dass diese Wechselwirkungen mit der Auswahl einer bestimmten Exosomkonformation (Zustand A im Energiediagramm) einhergehen.

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