ABSTRACT
An important step in the process of protein research by NMR is the assignment of chemical shifts. In the coat protein of IKe bacteriophage, there are 53 residues making up a long helix resulting in relatively high spectral ambiguity. Assignment thus requires the collection of a set of three-dimensional (3D) experiments and the preparation of sparsely labeled samples. Increasing the dimensionality can facilitate fast and reliable assignment of IKe and of larger proteins. Recent progress in nonuniform sampling techniques made the application of multidimensional NMR solid-state experiments beyond 3D more practical. 4D 1 H-detected experiments have been demonstrated in high-fields and at spinning speeds of 60 kHz and higher but are not practical at spinning speeds of 10-20 kHz for fully protonated proteins. Here, we demonstrate the applicability of a nonuniformly sampled 4D 13 C/15 N-only correlation experiment performed at a moderate field of 14.1 T, which can incorporate sufficiently long acquisition periods in all dimensions. We show how a single CANCOCX experiment, supported by several 2D carbon-based correlation experiments, is utilized for the assignment of heteronuclei in the coat protein of the IKe bacteriophage. One sparsely labeled sample was used to validate sidechain assignment of several hydrophobic-residue sidechains. A comparison to solution NMR studies of isolated IKe coat proteins embedded in micelles points to key residues involved in structural rearrangement of the capsid upon assembly of the virus. The benefits of 4D to a quicker assignment are discussed, and the method may prove useful for studying proteins at relatively low fields.