PHRONESIS: A One‐Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid‐State NMR Spectroscopy

Solid‐state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane‐bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, (1)H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance‐specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi‐acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D (1)H‐detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de‐excitation of specific resonances were achieved by high‐fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U‐(13)C,(15)N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel (1)H‐detected multi‐acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems.