Intrinsically disordered proteins (IDPs) carry out many biological functions. They lack a stable three-dimensional structure, but rather adopt many different conformations in dynamic equilibrium. The interplay between local dynamics and global rearrangements is key for their function. In IDPs, proline residues are significantly enriched. Given their unique physicochemical and structural properties, a more detailed understanding of their potential role in stabilizing partially folded states in IDPs is highly desirable. Nuclear magnetic resonance (NMR) spectroscopy, and in particular 13 C-detected NMR, is especially suitable to address these questions. We applied a 13 C-detected strategy to study Osteopontin, a largely disordered IDP with a central compact region. By employing the exquisite sensitivity and spectral resolution of these novel techniques we gained unprecedented insight into cis-Pro populations, their local structural dynamics and their role in mediating long-range contacts. Our findings clearly call for a reassessment of the structural and functional role of proline residues in IDPs. The emerging picture shows that proline residues have ambivalent structural roles. They are not simply disorder promoters but rather can, depending on the primary sequence context, act as nucleation sites for structural compaction in IDPs. These unexpected features provide a versatile mechanistic toolbox to enrich the conformational ensembles of IDPs with specific features for adapting to changing molecular and cellular environments.

While CH–π interactions with target proteins are crucial determinants for the affinity of arguably every drug molecule, no method exists to directly measure the strength of individual CH–π interactions in drug–protein complexes. Herein, we present a fast and reliable methodology called PI (π interactions) by NMR, which can differentiate the strength of protein–ligand CH–π interactions in solution. By combining selective amino‐acid side‐chain labeling with 1H‐13C NMR, we are able to identify specific protein protons of side‐chains engaged in CH–π interactions with aromatic ring systems of a ligand, based solely on 1H chemical‐shift values of the interacting protein aromatic ring protons. The information encoded in the chemical shifts induced by such interactions serves as a proxy for the strength of each individual CH–π interaction. PI by NMR changes the paradigm by which chemists can optimize the potency of drug candidates: direct determination of individual π interactions rather than averaged measures of all interactions.