High-quality ¹³C-detected structural analysis of mass-limited amyloid samples using a CPMAS CryoProbe and moderate...

Solid-state NMR (SSNMR) of biomolecules typically requires several milligrams of sample to achieve sufficient sensitivity for multidimensional experiments, especially when relying on ¹³C detection. Recent developments in fast magic-angle spinning (MAS) and ¹H-detected methods have enabled the use of submilligram samples in reduced-diameter rotors, but these approaches demand advanced hardware and often suffer from limited ¹H chemical shift dispersion. Here, we demonstrate that a CPMAS CryoProbe enables the acquisition of high-quality ¹³C-detected 2D and 3D spectra from just ∼1.5 mg of uniformly labeled amyloid fibrils packed in a standard 3.2 mm rotor. As a proof of concept, we apply this approach to RIPK3, a key protein in immune signaling that forms functional amyloid assemblies. Using standard 3D experiments (NCACX and NCOCX), we obtain ¹³C and ¹⁵N backbone assignments and secondary structure information, despite the limited sample quantity and the use of only moderate magnetic fields. These findings highlight the potential of CPMAS CryoProbes to shift the paradigm in mass-limited SSNMR studies, from relying exclusively on ¹H-detection and fast MAS to reembracing ¹³C-detected strategies.