Simulation at a certain pH

Dear all,
For pH-dependant simulation, I use H++ server and by -flag aminoacids I protonate proteins.
But, I am not sure if I need to neutralize the system.
In addition, I did simulations of protein/acetic acid (pH ~ 3) and protein/aqueous ethanol. Experimentally, it should have shown that acetic acid unfolded proteins more than ethanol (based on Radius of gyraton), but it did not happen. Could you please give me some advice about the neutralizing system in pH-dependent simulation and the Rg results of acetic acid and ethanol?
Many thanks in advance,


currently in GROMACS you can run only at fix protonation state.
Knowing that once you have determined the protonation state of your residues (according to the desired pH), the system need to be neutralize with counter ions (in particular if you use PME to describe long range interaction).
Best regards