Super-resolution triple-resonance NMR spectroscopy for the sequential assignment of proteins
2025-08-15
To study the structure and dynamics of proteins by nuclear magnetic resonance (NMR), sequence-specific assignment is needed, which can be obtained by acquiring and analyzing multiple triple-resonance experiments with the three-dimensional TROSY-HNCA, the most sensitive stand-alone experiment with which sequential assignment is, in principle, possible. However, gaining an unambiguous assignment solely from this spectrum is generally not possible because amino acid–type information cannot be gleaned only from the 13 C α shifts and the low resolution in the 13 C dimension, which is limited by the homonuclear coupling of the 13 C α and 13 C β nuclei. Here, super-resolution NMR is applied to the TROSY-HNCA and HNcoCA experiments, yielding pseudo-decoupling, which results in a four- to fivefold resolution enhancement in the 13 C dimension, essential for the assignment, which allows for straightforward assignment of proteins as large as 500 residues based on simulations.