4.3. Conformational Analysis

The molecular dynamic simulations are performed with Gromacs 4.6.5 [35]. The interactions of the molecule are modelled with the GAFF force field [12,36] in explicit water. After energy minimization and water thermalization, we performed 50 ns of simulation at 300 K, recording 1000 conformations to be analyzed.

The conformational ensembles of the molecules are built on the basis of the NOE intensities with the strategy described in [12], treating independently the α and β isoforms of the two compounds. An initial model for each of the four molecules was built within the GAFF force field [36]. Iterative MD simulations are performed with the Gromacs 4.5 package in implicit solvent, updating the torsional terms of the potential (except those affecting the amidic bonds) to minimize the χ² between the experimental and the back-calculated NOE intensities (cf. also Figure S8 in the Supplementary Materials). To avoid overfitting, the iterations are stopped when χ² = 1, that is, when the back-calculated NOE intensities are within the error bars of the data, obtained from the triplicate experiment.

The NOEs obtained from three independent experiments, together with the associated mean and standard deviations, are reported in Figure S3 in the Supplementary Materials The forward model to back-calculate the NOE intensities for each pair of hydrogen atoms at distance d from the ensemble of simulated conformations is:

where the angular bracket indicates the average over the simulated conformations, and I₀ is 18.2 for compound 1, and 3.58 for compound 2. This forward model holds [37], as the inter-atomic distance correlation time is slower than the rotational correlation time (cf. Figure S9).