Observation of intersubunit NOEs in a dimeric P22 Mnt repressor mutant by a time-shared [¹⁵N, ¹³C] double half-filter technique

A time-shared [¹⁵N, ¹³C] half-filter technique is presented, which can be used to study proton-proton NOEs between biomolecules. The filter is demonstrated in a 2D [¹⁵N, ¹³C] double filtered NOESY experiment of a dimeric Mnt repressor mutant consisting of completely [¹⁵N, ¹³C] labeled monomer and unlabeled monomer. The benefit of this combined [¹⁵N, ¹³C] half-filter is that a single NMR experiment can be designed that yields all NOE interactions between labeled and unlabeled protons ((¹³C, ¹⁴N/¹²C), (¹⁵N, ¹⁴N/¹²C), (¹²C, ¹⁵N/¹³C) and (¹⁴N, ¹⁵N/¹³C)) in the protein, where conventional half-filters would require at least three separate NMR experiments to obtain these NOEs. The intermonomer NOEs of the Mnt mutant confirmed the secondary structure of the DNA-binding domain as an antiparallel β ribbon, formed from an N-terminal segment contributed by each monomer. Moreover, several intersubunit NOEs were characterized in the C-terminal part of the Mnt mutant for which no structural data is available yet.