Determining the Importance of Hydrogen Bond Strength on the Mechanical Stability of Proteins

Proteins are only marginally stable at room temperature and can easily be unfolded when subjected to denaturants such as chemicals or mechanical forces. Despite this, proteins are able to maintain a unique 3D structure held together by interactions such as hydrogen bonds and hydrophobic interactions in physiological conditions. We have used single molecule force spectroscopy (SMFS) to investigate the effect of hydrogen bond strength on the mechanical resilience of two proteins: I27, from the giant muscle protein titin, and protein L, an immunoglobulin binding domain from bacterial cell wall. By picking up and stretching a single protein, the unfolding force needed to unravel it can be determined; this infers information about the molecular interactions that confer its stability and flexibility.

To directly test the importance of hydrogen bond strength, we have exploited the use of deuterium (D or 2H) and deuterium oxide (D2O). Deuterium has been shown to form strong hydrogen bonds than hydrogen (1.04 - 2.07 kT stronger). Protein engineering was used to create protonated and deuterated versions of the proteins and SMFS experiments have been completed on the proteins in both water (H2O) and D2O. Our single molecule studies indicate that the mechanical resilience of both proteins is sensitive to the hydrogen bond strength in both the protein and the solvent. Furthermore, the changes in the mechanical resilience of the protein is coupled with an changes in the spring constant of the protein chain. The changes observed in the properties of the proteins due isotopic substitution could aid differentiation between the interactions involved within the rate limiting step of mechanical unfolding. This study has illustrated the sensitivity of SMFS experiments to small changes in the strength of the interactions within proteins, and the importance of probing the net contribution of different interactions to protein stability.