Force Field Evaluation for an Intrinsically Disordered Domain: MD–NMR–FCS Benchmarking of Protein 4.1G Headpiece Ensembles

Intrinsically disordered proteins and regions (IDPs/IDRs) possess pronounced conformational heterogeneity, complicating their structural characterization. Integrating molecular dynamics (MD) simulations with experimental constraints is therefore indispensable for generating accurate conformational ensembles. A central factor limiting the accuracy of such simulations is the adequacy of force field parametrization for IDPs. In this study, we systematically characterized the intrinsically disordered headpiece (HP) domain of protein 4.1G by employing replica-exchange MD simulations alongside NMR spectroscopy and fluorescence correlation spectroscopy (FCS). Six force field/water model combinations were evaluated by benchmarking simulated ensembles against experimental NMR chemical shifts and hydrodynamic radii. Initiating simulations with a cis Ile78–Pro79 peptide bond markedly improved agreement with NMR chemical shifts. Of the tested combinations, C36IDPSFF/TIP3Pm and ff99SBdisp/TIP4P-disp showed better agreement with the available local NMR-derived descriptors. Global conformational analysis revealed distinct force field biases: ff14SB/TIP4P-D, ff99SB-ILDN/TIP3P, and ESFF1/TIP4P-D favored compact ensembles, whereas ff99SBdisp/TIP4P-disp produced the most expanded conformations. Conversion of the radius of gyration ( R g ) to hydrodynamic radius ( R H ) yielded values approaching the experimental measurement (8.20 Å). However, this conversion proved model-dependent, complicating a definitive ranking of force field performance. These results highlight the critical impact of force field choice on both local and global properties of IDPs/IDRs and emphasize the need for integrated, multimetric validation in the computational study of disordered proteins.