A novel high-dimensional NMR experiment for resolving protein backbone dihedral angle ambiguities.

Intrinsically disordered proteins (IDPs) are challenging established structural biology perception and urge a reassessment of the conventional understanding of the subtle interplay between protein structure and dynamics. Due to their importance in eukaryotic life and central role in protein interaction networks, IDP research is a fascinating and highly relevant research area in which NMR spectroscopy is destined to be a key player. The flexible nature of IDPs, as a result of the sampling of a vast conformational space, however, poses a tremendous scientific challenge, both technically and theoretically. Pronounced signal averaging results in narrow signal dispersion and requires higher dimensionality NMR techniques. Moreover, a fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution, where often the interpretation relies on the concept of 'residual structure' or 'conformational preference'. An important source of structural information is information-rich NMR experiments that probe protein backbone dihedral angles in a unique manner. Cross-correlated relaxation experiments have proven to fulfil this task as they provide unique information about protein backbones, particularly in IDPs. Here we present a novel cross-correlation experiment that utilizes non-uniform sampling detection schemes to resolve protein backbone dihedral ambiguities in IDPs. The sensitivity of this novel technique is illustrated with an application to the prototypical IDP [Formula: see text]-Synculein for which unexpected deviations from random-coil-like behaviour could be observed.

Protonation-dependent conformational variability of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity and undergo rearrangements of the time-averaged conformational ensemble on changes of environmental conditions (e.g., in ionic strength, pH, molecular crowding). In contrast to stably folded proteins, IDPs often form compact conformations at acidic pH. The biological relevance of this process was, for example, demonstrated by nuclear magnetic resonance studies of the aggregation prone (low pH) state of α-synuclein. In this study, we report a large-scale analysis of the pH dependence of disordered proteins using the recently developed meta-structure approach. The meta-structure analysis of a large set of IDPs revealed a significant tendency of IDPs to form α-helical secondary structure elements and to preferentially fold into more compact structures under acidic conditions. The predictive validity of this novel approach was demonstrated with applications to the tumor-suppressor BASP1 and the transcription factor Tcf4.

Cooperative unfolding of compact conformations of the intrinsically disordered protein osteopontin.

Intrinsically disordered proteins (IDPs) constitute a class of biologically active proteins that lack defined tertiary and often secondary structure. The IDP Osteopontin (OPN), a cytokine involved in metastasis of several types of cancer, is shown to simultaneously sample extended, random coil-like conformations and stable, cooperatively folded conformations. By a combination of two magnetic resonance methods, electron paramagnetic resonance and nuclear magnetic resonance spectroscopy, we demonstrate that the OPN ensemble exhibits not only characteristics of an extended and flexible polypeptide, as expected for an IDP, but also simultaneously those of globular proteins, in particular sigmoidal structural denaturation profiles. Both types of states, extended and cooperatively folded, are populated simultaneously by OPN in its apo state. The heterogeneity of the structural properties of IDPs is thus shown to even involve cooperative folding and unfolding events.