A single-molecule perspective on the role of solvent hydrogen bonds in protein folding and chemical reactions.

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 (H(2)O) with deuterium oxide (D(2)O). 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 H(2)O with D(2)O. We find that D(2)O molecules form an integral part of the unfolding transition structure of the immunoglobulin module of human cardiac titin, I27. Strikingly, we find that D(2)O is a worse solvent than H(2)O for the protein I27, in direct contrast with the behaviour of simple hydrocarbons. We measure the effect of substituting H(2)O with D(2)O on the force dependent rate of reduction of a disulphide bond engineered within a single protein. Altogether, these experiments provide new information on the nature of the underlying interactions in protein folding and chemical reactions and demonstrate the power of single-molecule techniques to identify the changes induced by a small change in hydrogen bond strength.