Experimentally, the free energy for the trans-to-cis isomerization barrier is 20.40 kcal•mol -1 , and a relative cis/trans ∆G is 0.57 kcal•mol -1 . While DFTB2-MIO/MM free energy profile recovers correctly the the position of the transition state and the relative energies of the cis and trans isomers (∆G = 0.52 kcal•mol -1 ), it underestimates the height of the barrier with a ∆G ‡ of 16.97 kcal•mol -1 . The DFTB2-OB2 profile correctly reproduces the relative energies of the cis and trans isomers although it slightly underestimates the cis/trans energy (∆G = 0.25 kcal•mol -1 ). The position of the transition state is correctly described, and the height of the trans-to-cis barrier is correctly reproduced albeit overestimated, with a ∆G ‡ of 21.89 kcal•mol -1 . Similar results have been obtained for the DFTB3-3OB profile, where the height of the barrier is overestimated with a ∆G ‡ of 21.91 kcal•mol -1 . However, contrarily to the two profiles computed at the DFTB2 level, the DFTB3 profile wrongly reproduces the relative energies of the cis and trans isomers, with the cis conformation predicted to be the more stable one (∆G = -1.07 kcal•mol -1 ). Based on these benchmarks, we decided to use DFTB2 in combination with OB2 parameters set as the default options for studying the peptide isomerization. In Fig. we reported the cis-to-trans isomerization free energy profiles in the succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF) peptide and proline, computed at the modified set of Amber99sb parameters and DFTB2-OB2 level of theory. The reaction barrier on peptide computed at the DFTB2 level (21.22 kcal•mol -1 ) shows a good agreement with the experimental value (19.26 kcal•mol -1 ). Although it is slightly overestimated, the position of the barrier and the relative energies of the cis/trans are correctly described.