Longitudinal relaxation properties of (1)H(N) and (1)H(α) determined by direct-detected (13)C NMR experiments to study intrinsically disordered proteins (IDPs).

Intrinsically disordered proteins (IDPs) are functional proteins containing large fragments characterized by high local mobility. Bioinformatic studies have suggested that a significant fraction (more than 30%) of eukaryotic proteins has disordered regions of more than 50 amino acids in length. Hence, NMR methods for the characterization of local compactness and solvent accessibility in such highly disordered proteins are of high importance. Among the available approaches, the HET-SOFAST/BEST experiments (Schanda et al., 2006, Rennella et al., 2014) provide semi-quantitative information by monitoring longitudinal (1)H relaxation of amide protons under different initial conditions. However, when approaching physiological sample conditions, the potential of these amide (1)H detected experiments is reduced due to rapid amide proton solvent exchange. (13)C direct detection methods therefore provide a valuable alternative thanks to a higher chemical shift dispersion and their intrinsic insensitivity toward solvent exchange. Here we present two sets of (13)C-detected experiments, which indirectly measure (1)H(N) and (1)H(α) inversion recovery profiles. The experiments consist of an initial spin inversion-recovery block optimized for selective manipulation of different types of proton spins followed by a CON read-out scheme. The proposed experiments were tested on human α-synuclein and ubiquitin, two representative examples of unfolded and folded proteins.