Solvent molecules bridge the mechanical unfolding transition state of a protein

We demonstrate a combination of single molecule force spectroscopy and solvent substitution that captures the presence of solvent molecules in the transition state structure. We measure the effect of solvent substitution on the rate of unfolding of the 127 titin module, placed under a constant stretching force. From the force dependency of the unfolding rate, we determine Delta x(u), the distance to the transition state. Unfolding the 127 protein in water gives a Delta x(u) = 2.5 angstrom, a distance that compares well to the size of a water molecule. Although the height of the activation energy barrier to unfolding is greatly increased in both glycerol and deuterium oxide solutions, Ox depends on the size of the solvent molecules. Upon replacement of water by increasing amounts of the larger glycerol molecules, Delta x(u) increases rapidly and plateaus at its maximum value of 4.4 angstrom. In contrast, replacement of water by the similarly sized deuterium oxide does not change the value of Ox. From these results we estimate that six to eight water molecules form part of the unfolding transition state structure of the 127 protein, and that the presence of just one glycerol molecule in the transition state is enough to lengthen Ox. Our results show that solvent composition is important for the mechanical function of proteins. Furthermore, given that solvent composition is actively regulated in vivo, it may represent an important modulatory pathway for the regulation of tissue elasticity and other important functions in cellular mechanics.