Super-resolution triple-resonance NMR spectroscopy for the sequential assignment of proteins.

Abstract

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 ¹³C^α shifts and the low resolution in the ¹³C dimension, which is limited by the homonuclear coupling of the ¹³C^α and ¹³C^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 ¹³C dimension, essential for the assignment, which allows for straightforward assignment of proteins as large as 500 residues based on simulations.