title: Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers.
abstract: Mitochondrial carriers, including uncoupling proteins, are unstable in detergents, which hampers structural and mechanistic studies. To investigate carrier stability, we have purified ligand-free carriers and assessed their stability with a fluorescence-based thermostability assay that monitors protein unfolding with a thiol-reactive dye. We find that mitochondrial carriers from both mesophilic and thermophilic organisms exhibit poor stability in mild detergents, indicating that instability is inherent to the protein family. Trends in the thermostability of yeast ADP/ATP carrier AAC2 and ovine uncoupling protein UCP1 allow optimal conditions for stability in detergents to be established but also provide mechanistic insights into the interactions of lipids, substrates, and inhibitors with these proteins. Both proteins exhibit similar stability profiles across various detergents, where stability increases with the size of the associated detergent micelle. Detailed analysis shows that lipids stabilize carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilizes by direct interactions as well. Cardiolipin reverses destabilizing effects of ADP and bongkrekic acid on AAC2 and enhances large stabilizing effects of carboxyatractyloside, revealing that this lipid interacts in the m-state and possibly other states of the transport cycle, despite being in a dynamic interface. Fatty acid activators destabilize UCP1 in a similar way, which can also be prevented by cardiolipin, indicating that they interact like transport substrates. Our controls show that carriers can be soluble but unfolded in some commonly used detergents, such as the zwitterionic Fos-choline-12, which emphasizes the need for simple validation assays like the one used here.
authors: From the Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors: Paul G
authors: PG
authors: Crichton
authors: From the Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors: Yang
authors: Y
authors: Lee
authors: From the Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors: Jonathan J
authors: JJ
authors: Ruprecht
authors: From the Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors: Elizabeth
authors: E
authors: Cerson
authors: From the Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors: Chancievan
authors: C
authors: Thangaratnarajah
authors: From the Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors: Martin S
authors: MS
authors: King
authors: From the Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom ek@mrc-mbu.cam.ac.uk.
authors: Edmund R S
authors: ER
authors: Kunji
mesh_terms: D002308:Cardiolipins / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D007473:Ion Channels / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008823:Micelles; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D012995:Solubility; D044366:Transition Temperature; D000071256:Uncoupling Protein 1
chemical_list: D002308:Cardiolipins; D003902:Detergents; D004791:Enzyme Inhibitors; D007473:Ion Channels; D008055:Lipids; D008823:Micelles; D024101:Mitochondrial Proteins; C112225:PET9 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C000604371:UCP1 protein, human; D000071256:Uncoupling Protein 1; D000226:Mitochondrial ADP, ATP Translocases
keywords: Adenine Nucleotide Translocase; Detergent Micelle; Differential Scanning Fluorimetry; Membrane Protein; Mitochondrial Transport; Protein Stability; Thermofluor Assay; Transporter; Uncoupling Protein
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