Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding
| Autor: | Justin M. Miller, Alyssa Pickens, Justin D. Marsee, Jessica Rodriguez, Sydney Moore |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Proteases Protein Denaturation Fluorophore medicine.medical_treatment lcsh:QR1-502 Biochemistry lcsh:Microbiology Anilino Naphthalenesulfonates Fluorescence Article 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine YME1 medicine Inner membrane Urea Molecular Biology chemistry.chemical_classification Protease proteostasis Nucleotides protein unfolding Tryptophan Molecular machine mitochondria 030104 developmental biology Proteostasis Enzyme chemistry Biophysics Unfolded protein response Solvents ATP-dependent protease AAA+ protease 030217 neurology & neurosurgery Peptide Hydrolases |
| Zdroj: | Biomolecules Volume 10 Issue 4 Biomolecules, Vol 10, Iss 656, p 656 (2020) |
| ISSN: | 2218-273X |
| Popis: | ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential protein substrates. Thus, these molecular machines must demonstrate greater stability than their substrates in order to ensure continued function in essential quality control networks. We report here a role for ATP in the stabilization of the inner membrane YME1L protease. Qualitative fluorescence data derived from protein unfolding experiments with urea reveal non-standard protein unfolding behavior that is dependent on [ATP]. Using multiple fluorophore systems, stopped-flow fluorescence experiments demonstrate a depletion of the native YME1L ensemble by urea-dependent unfolding and formation of a non-native conformation. Additional stopped-flow fluorescence experiments based on nucleotide binding and unfoldase activities predict that unfolding yields significant loss of active YME1L hexamers from the starting ensemble. Taken together, these data clearly define the stress limits of an important mitochondrial protease. |
| Databáze: | OpenAIRE |
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