15N-detected TROSY for 1H-15N heteronuclear correlation to study intrinsically disordered proteins: strategies to increase spectral quality

Abstract

Intrinsically disordered proteins and protein regions are central to many biological processes but difficult to characterize at atomic resolution. Nuclear magnetic resonance is particularly well-suited for providing structural and dynamical information on intrinsically disordered proteins, but existing NMR methodologies need to be constantly refined to provide greater sensitivity and resolution, particularly to capitalise on the potential of high magnetic fields to investigate large proteins. In this paper, we describe how 15N-detected 2D NMR experiments can be optimised for better performance. We show that using selective aliphatic 1H decoupling in N-TROSY type experiments results in significant increases in sensitivity and resolution for a prototypical intrinsically disordered protein, α-synuclein, as well as for a heterogeneous intrinsically disordered region of a large multidomain protein, CBP-ID4. We also investigated the performance of incorporating longitudinal relaxation enhancement in N-TROSY experiments, both with and without aliphatic 1H decoupling, and discussed the findings in light of the available information for the two systems.