Staphylokinase Displays Surprisingly Low Mechanical Stability

Single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations have revealed that shear topology is an important structural feature for mechanically stable proteins. Proteins containing a beta-grasp fold display the typical shear topology and are generally of significant mechanical stability. In an effort to experimentally identify mechanically strong proteins using single-molecule atomic force microscopy, we found that staphylokinase (SAK), which has a typical beta-grasp fold and was predicted to be mechanically stable in coarse-grained MD simulations, displays surprisingly low mechanical stability. At a pulling speed of 400 nm/s, SAK unfolds at similar to 60 pN, making it the mechanically weakest protein among the beta-grasp fold proteins that have been characterized experimentally. In contrast, its structural homologous protein streptokinase beta domain displays significant mechanical stability under the same experimental condition. Our results showed that the large malleability of native-state SAK is largely responsible for its low mechanical stability. The molecular origin of this large malleability of SAK remains unknown. Our results reveal a hidden complexity in protein mechanics and call for a detailed investigation into the molecular determinants of the protein mechanical malleability.