In the case of E18-C106, again a flattening in the ∆G buffer region and, evidently, non-sigmoidal individual site pK titration curves suggested a coupling between the residues (Figure ). Here, computing the adjusted pK a downshifts C106 from 12.18 ± 0.07 to 8.84 ± 0.04, bringing the estimate closer to the experimental value of 5.4 ± 0.2 (Figure ). However, this still leaves more than a 3 pK unit discrepancy between calculation and experiment. Previous work on homodimeric DJ-1 has revealed that two arginine residues (R48 and R28 from the other monomer) facilitate anion binding, which results in pK a elevation (Figure , right). In our simulations only positive counterions (i.e., no salt concentration) were present; however, through-space interactions as a result of a second arginine may also play a role in affecting the pK a . To this end, we probed the pK a of monomeric DJ-1. We found similar qualitative agreement in the curves between the dimeric and monomeric forms; however, rather than raising the pK a , the elimination of the second arginine shifted the pK a of C106 down to 6.78 ± 0.19 (Figure , left).