UC Berkeley

Changes in the cellular environment, such as the hundreds of possible posttranslational modifications, modulate protein energy landscapes to drive important biology, with consequences for signaling, allostery, and other vital processes. The effects of ubiquitination are particularly important because of their potential influence on degradation by the 26S proteasome. Specifically, proteasomal engagement requires unstructured initiation regions that many known proteasome substrates lack. This paradox raises the intriguing possibility that ubiquitin modification itself may induce local or global destabilization, thus revealing the requisite unstructured region for proteasome engagement. However, experimental determination has been hampered by difficulty in producing biophysical quantities of pure, native isopeptide-conjugated ubiquitinated samples. Further, any measured signal must be decoupled between ubiquitin and the substrate protein.

To assess the energetic effects of ubiquitination and how these manifest at the proteasome, we developed a generalizable strategy to create isopeptide-linked ubiquitin within structured regions of a protein. The effects on the energy landscape vary from negligible to dramatic, depending on the protein and site of ubiquitination. Ubiquitination at sensitive sites destabilizes the native structure and increases the rate of proteasomal degradation. Importantly, in well-folded proteins, ubiquitination can even induce the requisite unstructured regions needed for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory strategy for energy-dependent substrate degradation. We further characterize the biophysical mechanism of site-specific destabilization and discover distinct modes of destabilization and stabilizing interactions between ubiquitin and the substrate at different sites.