Sensitivity improvement for correlations involving arginine side-chain Nε/Hε resonances in multi-dimensional NMR experiments using broadband ¹⁵N 180° pulses

Abstract

Due to practical limitations in available ¹⁵N rf field strength, imperfections in ¹⁵N 180° pulses arising from off-resonance effects can result in significant sensitivity loss, even if the chemical shift offset is relatively small. Indeed, in multi-dimensional NMR experiments optimized for protein backbone amide groups, cross-peaks arising from the Arg guanidino ¹⁵Nε (~85 ppm) are highly attenuated by the presence of multiple INEPT transfer steps. To improve the sensitivity for correlations involving Arg Nε–Hε groups, we have incorporated ¹⁵N broadband 180° pulses into 3D ¹⁵N-separated NOE-HSQC and HNCACB experiments. Two ¹⁵N-WURST pulses incorporated at the INEPT transfer steps of the 3D ¹⁵N-separated NOE-HSQC pulse sequence resulted in a ~1.5-fold increase in sensitivity for the Arg Nε–Hε signals at 800 MHz. For the 3D HNCACB experiment, five ¹⁵N Abramovich-Vega pulses were incorporated for broadband inversion and refocusing, and the sensitivity of Arg¹Hε-¹⁵Nε-¹³Cγ/¹³Cδ correlation peaks was enhanced by a factor of ~1.7 at 500 MHz. These experiments eliminate the necessity for additional experiments to assign Arg ¹Hε and ¹⁵Nε resonances. In addition, the increased sensitivity afforded for the detection of NOE cross-peaks involving correlations with the ¹⁵Nε/¹Hε of Arg in 3D ¹⁵N-separated NOE experiments should prove to be very useful for structural analysis of interactions involving Arg side-chains.