We present an array of force spectroscopy experiments that aim to identify the role of solvent hydrogen bonds in protein folding and chemical reactions at the single-molecule level. In our experiments we control the strength of hydrogen bonds in the solvent environment by substituting water (H2O) with deuterium oxide (D2O). Using a combination of force protocols, we demonstrate that protein unfolding, protein collapse, protein folding and a chemical reaction are affected in different ways by substituting H2O with D2O. We find that D2O molecules form an integral part of the unfolding transition structure of the immunoglobulin module of human cardiac titin, I27. Strikingly, we find that D2O is a worse solvent.
