Stable and Minimum\nSize Solubilization of Membrane\nProteins with Cocktails of Phospholipid Analogues

Membrane proteins (MPs) play important roles in various\ncellular\nprocesses and are major targets for drugs. Solubilization of MPs is\noften needed for structural and biophysical studies. For high-resolution\nnuclear magnetic resonance measurements, there is a size limit of\nthe sample (<100 kDa), and a high thermal stability at an increased\ntemperature is required. Furthermore, lipid bilayer-like environments\nare desirable to preserve the native states of MPs. However, existing\nsolubilization techniques do not fulfill these requirements at the\nsame time. In this study, we combined two phospholipid analogues as\na solubilizer and stabilizer to isolate MPs. This method maintained\nbacteriorhodopsin (bR) extracted from purple membranes in its native\nstate for 7 d at 40 °C. The solubility was comparable to that\nof conventional detergents for MPs, and the thermal stability of the\nsolubilizate was the best among them. The increase in the molecular\nsize caused by the solubilization of bR was only 20 kDa, indicating\nthat 20 phospholipid analogue molecules were sufficient to solubilize\none bR molecule. <sup>15</sup>N–<sup>1</sup>H heteronuclear\nsingle quantum coherence spectra of solubilized <sup>2</sup>H- and <sup>15</sup>N-labeled bR gave ∼80% of the expected peaks. In addition,\nthe lysate of human neuropeptide Y<sub>2</sub> receptor-expressing\nmammalian cells exhibited ligand recognition for 7 d at 37 °C,\nsuggesting that this technique can be used for ligand screening. Moreover,\nthe structure of the single membrane-spanning M2 protein of the influenza\nA virus expressed in <i>Escherichia coli</i> was stably\nmaintained for 7 d at 40 °C. Thus, our method is promising for\nvarious MP studies.