RESUMO
Maximum entropy methods (MEMs) determine posterior distributions by combining experimental data with prior information. MEMs are frequently used to reconstruct conformational ensembles of molecular systems for experimental information and initial molecular ensembles. We performed time-resolved Förster resonance energy transfer (FRET) experiments to probe the interdye distance distributions of the lipase-specific foldase Lif in the apo state, which likely has highly flexible, disordered, and/or ordered structural elements. Distance distributions estimated from ensembles of molecular dynamics (MD) simulations serve as prior information, and FRET experiments, analyzed within a Bayesian framework to recover distance distributions, are used for optimization. We tested priors obtained by MD with different force fields (FFs) tailored to ordered (FF99SB, FF14SB, and FF19SB) and disordered proteins (IDPSFF and FF99SBdisp). We obtained five substantially different posterior ensembles. As in our FRET experiments the noise is characterized by photon counting statistics, for a validated dye model, MEM can quantify consistencies between experiment and prior or posterior ensembles. However, posterior populations of conformations are uncorrelated to structural similarities for individual structures selected from different prior ensembles. Therefore, we assessed MEM simulating varying priors in synthetic experiments with known target ensembles. We found that (i) the prior and experimental information must be carefully balanced for optimal posterior ensembles to minimize perturbations of populations by overfitting and (ii) only ensemble-integrated quantities like inter-residue distance distributions or density maps can be reliably obtained but not ensembles of atomistic structures. This is because MEM optimizes ensembles but not individual structures. This result for a highly flexible system suggests that structurally varying priors calculated from varying prior ensembles, e.g., generated with different FFs, may serve as an ad hoc estimate for MEM reconstruction robustness.
RESUMO
Molecular dynamics (MD) simulations of explicit representations of fluorescent dyes attached via a linker to a protein allow, e.g., probing commonly used approximations for dye localization and/or orientation or modeling Förster resonance energy transfer. However, setting up and performing such MD simulations with the AMBER suite of biomolecular simulation programs has remained challenging due to the unavailability of an easy-to-use set of parameters within AMBER. Here, we adapted the AMBER-DYES parameter set derived by Graen et al. [J. Chem. Theory Comput. 10, 5505 (2014)] into "AMBER-DYES in AMBER" to generate a force field applicable within AMBER for commonly used fluorescent dyes and linkers attached to a protein. In particular, the computationally efficient graphics processing unit (GPU) implementation of the AMBER MD engine can now be exploited to overcome sampling issues of dye movements. The implementation is compatible with state-of-the-art force fields such as GAFF, GAFF2, ff99SB, ff14SB, lipid17, and GLYCAM_06j, which allows simulating post-translationally modified proteins and/or protein-ligand complexes and/or proteins in membrane environments. It is applicable with frequently used water models such as TIP3P, TIP4P, TIP4P-Ew, and OPC. For ease of use, a LEaP-based workflow was created, which allows attaching (multiple) dye/linker combinations to a protein prior to further system preparation steps. Following the parameter development described by Graen et al. [J. Chem. Theory Comput. 10, 5505 (2014)] and the adaptation steps described here, AMBER-DYES in AMBER can be extended by additional linkers and fluorescent molecules.