Quantitative Integration of FRET and Molecular Dynamics for Modeling Flexible Peptides

Flexible protein regions, often enriched in glycine- and serine-rich segments, play a central role in biomolecular dynamics and function. The combination of time-resolved fluorescence resonance energy transfer (FRET) spectroscopy and molecular dynamics simulations provides a powerful framework to characterize these motions at atomic resolution. In this work, we investigate the conformational and kinetic properties of Trp-(GS)_n-Dbo and Trp-(PP)_n-Dbo peptides (n = 0, 1, 2, 3) in aqueous solution using microsecond-scale MD simulations, informed by an improved description of the Dbo-labeled aspartic residue compatible with the G54A7 force field. The simulations quantitatively reproduce experimental end-to-end distances derived from FRET measurements, with deviations below 5% for all (GS)_n peptides, and correctly capture the systematic relationship between chain flexibility and fluorophore separation. Analysis of looping kinetics further shows quantitative agreement with experimentally measured contact formation rates after viscosity correction, supporting a diffusion-controlled mechanism for intrachain contact formation. Together, these results establish a consistent, quantitative link between structural ensembles, dynamical observables, and FRET experiments, and provide benchmark data for modeling fluorophore-labeled peptides and intrinsically disordered protein segments.