Probing the atomic details of intrinsically disordered proteins is crucial to understanding their biological function and relation to pathogenesis. Although amide-detected NMR experiments are widely employed in protein studies, 3 J HNHα couplings between amide (1HN) and alpha (1Hα) protons impose an intrinsic limit on the achievable 1HN linewidth. Here, we present a homonuclear decoupling method that narrows the α-synuclein 1HN linewidths to 3–5 Hz. Tightly distributed 1 J CαHα coupling values were employed to generate homogeneous antiphase coherences of 2Hα z HN y and 4Hα(2) z Hα(3) z HN y for nonglycine and glycine residues, respectively, which were combined with their in-phase HN y counterparts to achieve homonuclear decoupling. By reducing the multiplet structure to a singlet, the width of the 1HN cross-peak was reduced by ∼3-fold in the 2D HSQC and 3D intra-HNCA spectra, and good spectral quality was achieved without the need for postprocessing.

Intrinsically disordered proteins (IDPs) play an important role in cell signaling, and NMR is well-suited to study conformational ensembles and dynamics of IDPs. However, the intrinsic flexibility of IDPs often results in severe spectral overlap, which hampers accurate NMR data analysis. By labeling the longitudinal spin order of an α proton (i.e., Hα z ) on multiple quantum coherences of backbone nuclei (e.g., N y C′ x Cα y ), we were able to apply pre-homonuclear decoupling (PHD) to transverse relaxation-optimized spectroscopy (TROSY). The proposed scheme provides ultrahigh resolution in both amide proton and nitrogen dimensions, as illustrated in the analysis of Tau and alpha-synuclein (α-Syn) proteins. The PHD-TROSY readout enabled complete backbone resonance assignment of α-Syn using a single 3D HNCA experiment performed on a 600 MHz NMR spectrometer.

Probing the protein surface accessibility of different residues is a powerful way of characterizing the overall conformation of intrinsically disordered proteins (IDPs). We present a two-dimensional (2D) time-resolved photo-CIDNP (TR-CIDNP) experiment suitable for IDP analysis. Pulse stretching of high-power laser pulses, band-selective decoupling of 13 C α , and simultaneous application of radiofrequency and laser pulses were implemented to quantitatively analyze the IDP surface at ultrahigh resolution. Comparative analysis with other methods that measure protein surface accessibility validated the newly developed method and emphasized the importance of dye charge in photo-CIDNP. Using the neutral riboflavin dye, surface accessibilities were measured to be nearly identical for the four Tyr residues of α-synuclein (α-Syn), whose 1 H α − 13 C α correlations were well-resolved in the 2D TR-CIDNP spectrum. Having confirmed the similarity between the time-resolved and steady-state photo-CIDNP results for α-Syn, we used the more sensitive latter method to show that divalent cations induce compaction of the C-terminal region and release of the Nterminal region of α-Syn. The photo-CIDNP method presented herein can be used as an orthogonal and independent method for investigating important biological processes associated with changes in the overall IDP conformation.

Enhancements in NMR sensitivity have been the main driving force to extend the boundaries of NMR applications. Recently, techniques to shift the thermally populated nuclear spin states are employed to gain high NMR signals. Here, we introduce a technique called photochemically induced dynamic nuclear polarization (photo-CIDNP) and discuss its progresses in enhancing the solution-state NMR sensitivity.