In order to test the potential influence of side chain ionization on folding, we repeated our simulations starting from the native NMR solution structure, which exhibits a salt bridge with a protonated His5 residue and the C-terminus Gly21 carboxylate. The imidazolium-bearing His5 could therefore attract the Gly21 carboxylate, contributing to the pre-lasso stability and might increase the hydrophilicity and solvation of the pre-ring. A 7.2 µs simulation resulted in a new free energy distribution for the protonated native sequence (Figure ). Within the 21 filtered ensemble, structures with Gly1N and Glu8Cδ distances ≤ 6 Å, the protonation of His5 decreases the relative probability of pre-lasso to pre-tadpole structures from 14.6% to only 0.8% (Table ). Despite this, the pre-tadpole population in the total trajectory is similar for neutral and protonated His5 (3.5% compared to 3.7%, respectively). Cumulatively, the protonation of His5 destabilizes the pre-lasso fold for native MccJ25 through increased solvation of the ring residues, which inhibits His5 Gly21 ring-tail interactions (further discussed below). Similarly, simulations starting from the pre-lasso with protonated His5 were run for MOD1 and MOD2 (Figure ). There was a general increase in the total number of filtered structures from the entire trajectory in both cases going from 3.7% (WT) to 10.7% (MOD1) and 9.9% (MOD2) with His5 protonated (Table ). From this set of structures, the percentage of prelassos in MOD1 significantly increases from 0.8% in the protonated native state to 45.6% (compared to 25.4% with neutral His5). On the other hand, MOD2 protonation decreased further from 3.7% (neutral His5) to 3.1% and promoted a pronounced increase of pre-tadpoles (6.5% of the total trajectory for neutral MOD2 to 9.6% with protonated MOD2).