TanGIBLE: A selective probe for evaluating hydrophobicity-exposed defective proteins in live cells.

The accumulation of defective polypeptides in cells is a major cause of various diseases. However, probing defective proteins is difficult because no currently available method can retrieve unstable defective translational products in a soluble state. To overcome this issue, there is a need for a molecular device specific to structurally defective polypeptides. In this study, we developed an artificial protein architecture comprising tandemly aligned BAG6 Domain I, a minimum substrate recognition platform responsible for protein quality control. This tandem-aligned entity shows enhanced affinity not only for model defective polypeptides but also for endogenous polyubiquitinated proteins, which are sensitive to translational inhibition. Mass-spectrometry analysis with this probe enabled the identification of endogenous defective proteins, including orphaned subunits derived from multiprotein complexes and misassembled transmembrane proteins. This probe is also useful for the real-time visualization of protein foci derived from defective polypeptides in stressed cells. Therefore, this "new molecular trap" is a versatile tool for evaluating currently "invisible" pools of defective polypeptides as tangible entities.