Methyl-TROSY NMR at 1.2 GHz: exploiting resolution and magnetic field alignment for structure and dynamics of large biomolecular assemblies
The range of protein assemblies that can be studied by NMR has greatly expanded both by increases in magnetic field strength and by development of new NMR approaches, such as methyl-group specific transverse relaxation optimized spectroscopy (methyl-TROSY). Recently, a new generation of NMR magnets operating at 1.2 GHz have become available, offering the highest resolution for biomolecular NMR. Here, we investigated the performance of methyl-TROSY NMR at 1.2 GHz in the characterization of the structure and dynamics of large biomolecular assemblies, using the nucleosome, a 200 kDa protein-DNA complex, as a test sample. We find that the increased resolution of the 1.2 GHz system allowed to resolve small asymmetries in sidechain conformations that remained hidden at lower magnetic field strengths. Increased CSA relaxation effectively increased 13C transverse relaxation rates by 20% at 1200 compared to 900 MHz, negatively impacting sensitivity. We further observed significant magnetic field alignment of the nucleosome at 1.2 GHz, giving rise to methyl 1H-13C residual dipolar couplings (RDCs). We show that these histone methyl group RDCs can be used to aid stereo-specific assignment and to determine the overall conformation of the nucleosomal DNA, revealing a significant unwrapping of the DNA from the histone core in solution. We expect that 1.2 GHz NMR will be very advantageous for studies of large biomolecular assemblies, and in particular for protein-DNA complexes.