Chen and Arnold (Proc, Natl. Acad. Sci. U.S.A. 1993, 90, 5618-5622) have generated a 10 amino acid mutant (PC3) of subtilisin E (SE) that has enhanced activity in mixed DMF/water solvent systems. Through the use of molecular dynamics simulations on both SE and PC3 in water, DMF, and DMF/water (PC3 only) solvent systems, we have provided insights into how nonaqueous solvents affect protein structure and dynamics. On the basis of the observations reported herein, we propose that the PC3 mutant protein is more compatible with DMF as solvent than is the native SE protein. The concept of solvent compatibility embodies the ideas that in order for a protein to be active in organic solvents it must be able to retain its overall shape and that it must not become too rigid such that catalytic activity is compromised. Moreover, neither should the active site region be obstructed by conformational changes that block or structurally alter the active site nor should the active site binding pocket be blocked by solvent molecules (i.e., solvent inhibition). Attempts to determine how each individual amino acid substitution might cause these effects met with mixed results. Clearly, in the present case the individual mutations synergistically lead to the alteration in function through numerous subtle local changes in the structure and dynamics of the protein. Nonetheless, from the simulations, we were able to make some predictions regarding how a protein might be stabilized in a nonaqueous solvent environment.