QEBSS: Quality Evaluation Based Simulation Selection for analysis of conformational ensembles and dynamics of multidomain proteins

Multi-domain proteins, comprising both folded and intrinsically disordered regions, play pivotal roles in various biological processes, yet characterizing their conformational ensembles and dynamics presents significant challenges. Here we address this challenge by introducing the Quality Evaluation Based Simulation Selection (QEBSS) protocol that enables detailed interpretation of conformational ensembles and dynamics of multi-domain proteins using a combination of MD simulations and protein backbone 15N T1 and T2 spin relaxation times and hetNOE values from NMR experiments. We demonstrate the practical advantage of QEBSS by solving conformational ensembles and dynamics of four flexible multi-domain proteins: calmodulin, EN2, MANF and CDNF. These proteins bear important biological functions but their mechanistic understanding is limited due to challenges in characterization of flexible multi-domain proteins. Our findings reveal new insights on conformational landscapes and dynamics of these proteins, shining light also on their biological functions. QEBSS provides a quantitative quality evaluation of simulations offering a systematic approach to resolve conformational ensembles and dynamics of multi-domain proteins with heterogeneous dynamics. Because such proteins play important roles in wide range of systems with relevance in biology, biotechnology and material sciences, we anticipate QEBSS to benefit wide range of fields ranging from drug design to development of novel materials.