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 mechan... More
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 stabilise carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilises by direct interactions as well. Cardiolipin reverses destabilising effects of ADP and bongkrekic acid on AAC2 and enhances large stabilising 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 destabilise 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 emphasises the need for simple validation assays like the one used here.
