The key event in force-induced unfolding of titin's immunoglobulin domains

Steered molecular dynamics simulation of force-induced titin immunoglobulin domain 127 unfolding led to the discovery of a significant potential energ y barrier at an extension of similar to 14 Angstrom on the unfolding pathwa y that protects the domain against stretching. Previous simulations showed that this barrier is due to the concurrent breaking of six interstrand hydr ogen bonds (H-bonds) between beta-strands A' and G that is preceded by the breaking of two to three hydrogen bonds between strands A and B, the latter leading to an unfolding intermediate. The simulation results are supported by Angstrom-resolution atomic force microscopy data. Here we perform a str uctural and energetic analysis of the H-bonds breaking. It is confirmed tha t H-bonds between strands A and B break rapidly. However, the breaking of t he H-bond between strands A' and G needs to be assisted by fluctuations of water molecules. In nanosecond simulations, water molecules are found to re peatedly interact with the protein backbone atoms, weakening individual int erstrand H-bonds until all six A'-G H-bonds break simultaneously under the influence of external stretching forces. Only when those bonds are broken c an the generic unfolding take place, which involves hydrophobic interaction s of the protein core and exerts weaker resistance against stretching than the key event.