Removing water from an EcoRI-noncognate DNA complex with osmotic stress

We recently showed that a nonspecific complex of the restriction nuclease E coRI with poly (dI-dC) sequesters significantly more water at the protein-D NA interface than the complex with the specific recognition sequence. The n onspecific complex seems to retain almost a full hydration layer at the int erface. We now find that at low osmotic pressures a complex of the restrict ion nuclease EcoRI with a DNA sequence that differs by only one base pair f rom the recognition site (a 'star' sequence) sequesters about 70 waters mor e than the specific one, a value virtually indistinguishable from nonspecif ic DNA. Unlike complexes with oligo (dI-dC) or with a sequence that differs by two base pairs from the recognition sequence, however, much of the wate r in the 'star' sequence complex is removed at high osmotic pressures. The energy of removing this water can be calculated simply from the osmotic pre ssure work done on the complex. The ability to measure not only the changes in water sequestered by DNA-protein complexes for different sequences, but also the work necessary to remove this water is a potentially powerful new tool for coupling inferred structural changes and thermodynamics.