Multi-dimensional NMR without coherence transfer: minimizing losses in large systems

Most multi-dimensional solution NMR experiments connect one dimension to another using coherence transfer steps that involve evolution under scalar couplings. While experiments of this type have been a boon to biomolecular NMR the need to work on ever larger systems pushes the limits of these procedures. Spin relaxation during transfer periods for even the most efficient ¹⁵N–¹H HSQC experiments can result in more than an order of magnitude loss in sensitivity for molecules in the 100 kDa range. A relatively unexploited approach to preventing signal loss is to avoid coherence transfer steps entirely. Here we describe a scheme for multi-dimensional NMR spectroscopy that relies on direct frequency encoding of a second dimension by multi-frequency decoupling during acquisition, a technique that we call MD-DIRECT. A substantial improvement in sensitivity of ¹⁵N–¹H correlation spectra is illustrated with application to the 21 kDa ADP ribosylation factor (ARF) labeled with ¹⁵N in all alanine residues. Operation at 4 °C mimics observation of a 50 kDa protein at 35 °C.