Analysis of a water mediated protein-protein interactions within RNase T1

Buried and well-ordered solvent molecules are an integral part of each fold ed protein. For a few individual water molecules, the exchange kinetics wit h solvent have been described in great detail. So far, little is known abou t the energetics of this exchange process. Here, we present an experimental approach to investigate water-mediated intramolecular protein-protein inte ractions by use of double mutant cycles. As a first example, we analyzed th e interdependence of the contribution of two side chains (Asn9 and Thr93) t o the conformational stability of RNase T1. Ln the folded state, both side chains are involved in the "solvation" of the same water molecule WAT1. The coupling of the contributions of Asn9 and Thr93 to the conformational stab ility of RNase T1 was measured by urea unfolding and differential scanning calorimetry. The structural integrity of each mutant was analyzed by X-ray crystallography. We find that the effects of the Asn9Ala and the Thr93Ala m utations on the conformational stability are additive in the corresponding double mutant. We conclude that the foe energy of the WAT1 mediated intramo lecular protein-protein interaction in the folded state is very similar to solvent mediated protein-protein interaction in the unfolded state.