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A computational solution to the protein folding problem is the holy grail of biomolecular simulation and of the corresponding force fields. The complexity of the systems used for folding simulations precludes a direct feedback between the simulations and the force fields, thus necessitating the study of simpler systems with sufficient experimental data to allow force field optimization and validation. Recent studies on short polyalanine peptides of increasing length (up to penta-alanine) indicated the presence of a systematic deviation between the experimental (NMR-derived) J-couplings and the great majority of biomolecular force fields, with the χ(2) values for even the best-performing force fields being in the 1.4-1.8 range. Here we show that by increasing the number of residues to seven and by achieving convergence through an increase of the simulation time to 2 μs, we can identify one force field (the AMBER99SB force field, out of the three force fields studied) which when compared with the experimental J-coupling data (and for a specific set of Karplus equation parameters and estimated J-coupling errors previously used in the literature) gave a value of χ(2) = 0.99, indicating that full statistical consistency between experiment and simulation is feasible. However, and as a detailed analysis of the effects of estimated errors shows, the χ(2) values may be unsuitable as indicators of the goodness of fit of the various biomolecular force fields.

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Short peptides serve as minimal model systems to decipher the determinants of foldability due to their simplicity arising from their smaller size, their ability to echo protein-like structural characteristics, and their direct implication in force field validation. Here, we describe an effort to identify small peptides that can still form stable structures in aqueous solutions. We followed the in silico folding of a selected set of 8640 tryptophan-containing tetra- and pentapeptides through 15 210 molecular dynamics simulations amounting to a total of 272.46 μs using explicit representation of the solute and full treatment of the electrostatics. The evaluation and sorting of peptides is achieved through scoring functions, which include terms based on interatomic vector distances, atomic fluctuations, and rmsd matrices between successive frames of a trajectory. Highly scored peptides are studied further via successive simulation rounds of increasing simulation length and using different empirical force fields. Our method suggested only a handful of peptides with strong foldability prognosis. The discrepancies between the predictions of the various force fields for such short sequences are also extensively discussed. We conclude that the vast majority of such short peptides do not adopt significantly stable structures in water solutions, at least based on our computational predictions. The present work can be utilized in the rational design and engineering of bioactive peptides with desired molecular properties.

FMS‐like tyrosine kinase 3 (FLT3) has been found to be mutated in ~ 30% of acute myeloid leukaemia patients. Small‐molecule inhibitors targeting FLT3 that are currently approved or still undergoing clinical trials are subject to drug resistance due to FLT3 mutations. How these mutations lead to drug resistance is hitherto poorly understood. Herein, we studied the molecular mechanism of the drug resistance mutations D835N, Y842S and M664I, which confer resistance against the most advanced inhibitors, quizartinib and PLX3397 (pexidartinib), using enzyme kinetics and computer simulations. In vitro kinase assays were performed to measure the comparative catalytic activity of the native protein and the mutants, using a bacterial expression system developed to this aim. Our results reveal that the differential drug sensitivity profiles can be rationalised by the dynamics of the protein–drug interactions and perturbation of the intraprotein contacts upon mutations. Drug binding induced a single conformation in the native protein, whereas multiple conformations were observed otherwise (in the mutants or in the absence of drugs). The end‐point kinetics measurements indicated that the three resistant mutants conferred catalytic activity that is at least as high as that of the reference without such mutations. Overall, our calculations and measurements suggest that the structural dynamics of the drug‐resistant mutants that affect the active state and the increased conformational freedom of the remaining inactive drug‐bound population are the two major factors that contribute to drug resistance in FLT3 harbouring cancer cells. Our results explain the mechanism of drug resistance mutations and can aid to the design of more effective tyrosine kinase inhibitors.