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

Abstract 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 introduce the Quality Evaluation Based Simulation Selection (QEBSS) protocol to address this challenge by systematically selecting ensembles from molecular dynamics (MD) simulation data that enable detailed interpretation of nuclear magnetic resonance (NMR) experiments. QEBSS is based on quality evaluation of large number of MD simulation trajectories against protein backbone 15N T1 and T2 spin relaxation times and hetNOE values from NMR experiments, and concomitant selection of simulations that form the best ensemble for the interpretation of experiments. We demonstrate the practical advantage of QEBSS by solving conformational ensembles and dynamics of four flexible multi-domain proteins: calmodulin, CDNF, MANF, and EN2. These proteins bear important biological functions but their mechanistic understanding is limited partially 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 offers 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.