Efficient high-field MAS-DNP NMR reveals hidden side chains in amyloid fibrils

It has been widely observed in bioNMR experiments that many biological molecules contain flexible parts or side chains that do not yield easily observable NMR signals in room-temperature experiments. The reasons for the missing peaks could be because the flexible regions might exhibit unfavorable dynamics that interfere with NMR experiments, which result in low NMR intensity below the noise floor. To circumvent this issue, we exploit a hyperpolarization technique known as dynamic nuclear polarization (DNP), which is usually performed at low temperatures for optimal performances. We have also compared 1H enhancements for amyloid fibrils doped with the SNAPol-1 and M-Tinypol radicals, and the 1H DNP spectrum demonstrates a much higher ε~30 for the SNAPol-1 radical than ε~10 for the TinyPol-doped sample. By combining the sensitivity gain bestowed by efficient DNP polarizing agent (SNAPol-1), the freezing of local motions at cryogenic temperature (~ 100 K), and high NMR resolution at high magnetic field (18.8 T), we have successfully recorded an unprecedented enhancement factor of ~50 on amyloid fibrils (NWD2) in magic-angle spinning (MAS) DNP experiments at a high magnetic field of 18.8 T. Moreover, multidimensional MAS NMR experiments have revealed NMR signals of flexible side chains that were previously inaccessible at conventional room-temperature experiments. We also demonstrate that sensitivity-enhanced 2D 15N-13C correlation experiments can be achieved in ~ 2 hours. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains otherwise hidden at room temperature.