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Chemical shift assignment is vital for nuclear magnetic resonance (NMR)-based studies of protein structures, dynamics, and interactions, providing crucial atomic-level insight. However, obtaining chemical shift assignments is labor intensive and requires extensive measurement time. To address this limitation, we previously proposed ARTINA, a deep learning method for automatic assignment of two-dimensional (2D)-4D NMR spectra. Here, we present an integrative approach that combines ARTINAwith AlphaFold and UCBShift, enabling chemical shift assignment with reduced experimental data, increased accuracy, and enhanced robustness for larger systems, as presented in a comprehensive study with more than 5000 automated assignment calculations on 89 proteins. We demonstrate that five 3D spectra yield more accurate assignments (92.59%) than pure ARTINA runs using all experimentally available NMR data (on average 10 3D spectra per protein, 91.37%), considerably reducing the required measurement time. We also showcase automated assignments of only 15Nlabeled samples, and report improved assignment accuracy in larger synthetic systems of up to 500 residues. [ABSTRACT FROM AUTHOR]

In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity "Cambrian-like" [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger "Ordovician-Silurian" fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.