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Abstract

Various processes in the living cell are regulated by very large protein machineries with molecular weights in the megadalton regime. However, structural investigations of these large systems by solution-state NMR are challenging due to their slow rotational diffusion. Here we describe an approach, which opens new perspectives in the investigation of soluble supramolecular modules by magic-angle spinning (MAS) NMR. The absence of rotational diffusion in MAS-induced protein sediments allows typical solid-state NMR techniques without the need of invasive precipitation or crystallization procedures. The combination with protein deuteration, proton-detection and paramagnetic relaxation enhancement enabled us to observe and to assign backbone amide resonances of a 20S proteasome assembly with a molecular weight of 1.1 MDa. Similarly, we used the approach to characterize the polydisperse and highly dynamic small heat-shock protein αB-crystallin (600 kDa) with respect to its copper-dependent chaperone activity and its mechanism of binding destabilized substrate proteins.