Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-beta arrangement rich in beta-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (similar to 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (>= 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of epsilon similar to 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with epsilon similar to 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.

Automated summary

High-field dynamic nuclear polarization (DNP) technique performed at low temperatures can enhance the structural investigation of amyloid fibrils by slowing down protein dynamics and improving the sensitivity of NMR signals, allowing the detection of flexible side chains that were previously inaccessible.