Abstract
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.
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Data availability
The data that support the findings of this study are available within the manuscript and its supplementary information or from the corresponding author upon reasonable request. All constructs generated for this study are also available from the corresponding author upon reasonable request. Source data for Figs. 2–4 are presented with the paper.
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Acknowledgements
We thank all members of the Marqusee and Martin laboratories for helpful discussions. We also thank B. Maguire and K. Dong for assistance with protein purification and troubleshooting expertise. We acknowledge support from the US National Institutes of Health: grant nos. R01-GM050945 (S.M.) and R01-GM094497 (A.M.). S.M. is a Chan Zuckerberg Biohub investigator. A.M. is an investigator of the Howard Hughes Medical Institute.
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E.C.C. and E.R.G. performed the experiments and analyzed data. E.C.C., E.R.G., A.M. and S.M. contributed to experimental design, data interpretation and manuscript preparation.
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Full uncropped gels from Fig. 2
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Carroll, E.C., Greene, E.R., Martin, A. et al. Site-specific ubiquitination affects protein energetics and proteasomal degradation. Nat Chem Biol 16, 866–875 (2020). https://doi.org/10.1038/s41589-020-0556-3
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DOI: https://doi.org/10.1038/s41589-020-0556-3
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