Backbone rigidity of disordered protein linkers from NMR experiments and MD simulations

Disordered protein linkers are essential for multidomain protein function and engineering, but quantitative methods for their biophysical characterization remain limited. We combined NMR experiments with molecular dynamics simulations to demonstrate that protein backbone 15N spin relaxation times correlates with backbone rigidities in short disordered linkers. Using a tailored version of the Quality Evaluation Based Simulation Selection (QEBSS) framework, we characterized four model peptides: (GGS)3, (GPS)3, K(AP)5K, and KKEEVKKEEV-(PK)7KEEVKKEEVKK, representing common natural and engineered linker repeats. Glycine-rich sequences showed slight looping tendencies, while proline-containing sequences adopted extended conformations with increased approximate persistence lengths and slower dynamics. Notably, sodium and calcium binding to charged peptides minimally affected rigidity, indicating electrostatics don't dominate linker stiffness. This integrated approach provides quantitative insights into disordered linker properties and MD simulation accuracy, offering biophysical understanding for protein design and machine learning model development.