Development of ClpXP as a tool for investigating the mechanical properties of biomolecules applied to polyglutamine repeat proteins

It has become increasingly apparent that mechanical force plays an important role in biology. Biophysical techniques such as optical tweezers and atomic force microscopy (AFM) have allowed the investigation of mechanical stability at the level of a single protein molecule. However, despite the increasing sensitivity of these techniques, it is still difficult to mimic precisely the geometry of extension, forces and loading rates applied in vivo. Here we have developed a technique using a bacterial proteasome, ClpXP, which allowed the investigation of the mechanical stability of proteins at more biologically relevant forces and loading rates. It was demonstrated that various degradation signals can be used to target the proteins under investigation to ClpXP where they were unfolded, translocated and degraded. Several ClpX variants were investigated and an assay developed using a pseudohexameric ClpX variant that allowed robust degradation of several proteins. This assay was used to investigate the properties of a protein containing a 30 polyglutamine repeat sequence. Previous studies of polyglutamine repeats, using AFM, have shown that it may have interesting mechanical properties: it has either extreme mechanical strength, or access to a conformation which has a high mechanical strength. It was shown that this protein can be completely degraded using ClpXP without any intermediate product, and without reducing the degradation rate compared to a control protein without a polyglutamine repeat. This demonstrates that this assay can be used to investigate proteins whose mechanical properties are of interest and that the loading rates and application of force applied by this assay differ enough from those of AFM that different and more biologically relevant results can be obtained.