Heteronuclear NMR spectroscopy for lysine NH₃ groups in proteins: Unique effect of water exchange on ¹⁵N transverse relaxation

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH₃ groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH ₃ groups, with ¹⁵N chemical shifts around ∼33 ppm at pH 5.8 and 35 °C. Measurement of water-exchange rates and various types of ¹⁵N transverse relaxation rates for these NH₃ groups, reveals that rapid water exchange dominates the ¹⁵N relaxation for antiphase coherence with respect to ¹H through scalar relaxation of the second kind. As a consequence of this phenomenon, ¹⁵N line shapes of NH₃ signals in a conventional ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D ¹H-¹⁵N correlation experiment that exclusively observes in-phase ¹⁵N transverse coherence (termed HISQC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t₁ (¹⁵N) time domain and as a result exhibits strikingly sharper ¹⁵N line shapes and higher intensities for NH₃ cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between ¹⁵Nζ and ¹³C_ε (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase ¹⁵N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH ₃ cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH₃ groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH₃ groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer.