As a fist step, we optimized the amide ! dihedral force field parameters, in order to obtain a trans-to-cis isomerization free energy profile consistent with the available experimental data (see extended details in the Methods section in the SI). The trans-to-cis isomerization of ! angles consists of a local conformation change that is often compensated by local variations of the backbone angles and of the residues flanking the amide. In order to asses how the free energy profile for the isomerization of ! is affected by the torsion of and , we selected these 3 dihedral angles as our CVs and ran WT-MetaD simulations activating/biasing the trans-to-cis transition of 2 (i.e., the torsion around the amide bond) in different conditions (Figure ). Preliminary WT-MetaD simulations showed that the free energy profile of the ! isomerization is not particularly influenced by the and torsions. Well-converged WT-MetaD runs allowed us to reconstruct the differences in free energies between the conformers ( G) and estimate the free energy barriers (to this end we used infrequent WT-MetaD simulations, as recrossing WT-MetaD simulations may underestimate the barrier heightssee extended Methods for details).