DIADECOMP: A new approach to analyze decompositions from projection spectroscopy

We demonstrate for the first time a complete small protein characterization with the projection-decomposition approach, including full assignments as well as determination of the 3D fold. In TOCSY- and NOESY-type 4D experiments, pairing of signals from hydrogens and from their respective heavy atoms in decompositions represents a new problem. An approach, referred to as "DIADECOMP" (diagonal decomposition), is introduced to solve this problem; it consists of two separate decompositions of the input projections, differing in a 45° rotation of the spectral axes. While DIADECOMP requires a somewhat complex formulation, in practice it results in observing signals in the rotated decompositions that correspond to sums or differences of frequencies. When applied to a small protein, human defensin β6, the analysis of a HCC(CO)NH-TOCSY with DIADECOMP results in largely unambiguous assignments of the aliphatic side chain groups. Furthermore, DIADECOMP applied to a ¹⁵N-HSQC-NOESY-¹⁵N-HSQC provides all expected short distances between amide groups (defined as all H_N-H_N distances <3.5Å in a reference structure). It is worth noting that short H_N-H_N distances unambiguously define α-helices, the alignment of β-strands in sheets, as well as the presence of β-bulges. This approach of using a minimal amount of NMR data, namely four projection experiments recorded in ∼2.5days, resulted for the human defensin β6 in complete assignments and a backbone fold with a RMSD of the non-flexible structure of 0.6Å. Uniqueness of decompositions specifically from TOCSY- and NOESY-type 4D experiments is discussed.