Changes in the cellular environment 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. Moreover, proteasomal engagement requires unstructured initiation regions that many known proteasome substrates lack. To assess the energetic effects of ubiquitination and how these manifest at the proteasome, we developed a generalizable strategy to produce 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. 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 mechanism for energy-dependent substrate degradation.

细胞环境的变化调节蛋白质能量景观以驱动重要的生物学，并对信号传导、变构和其他重要过程产生影响。泛素化的影响特别重要，因为它们对 26S 蛋白酶体的降解具有潜在影响。此外，蛋白酶体参与需要许多已知的蛋白酶体底物缺乏的非结构化起始区域。为了评估泛素化的能量效应以及这些效应如何在蛋白酶体中表现出来，我们开发了一种可推广的策略，在蛋白质的结构化区域内产生与异肽连接的泛素。根据蛋白质和泛素化位点，对能源格局的影响从可忽略不计到显着变化。敏感位点的泛素化会破坏天然结构并增加蛋白酶体降解的速率。在折叠良好的蛋白质中，泛素化甚至可以诱导蛋白酶体参与所需的非结构化区域。我们的结果表明位点特异性泛素化作为能量依赖性底物降解的潜在调节机制的生物物理作用。