Stable and Minimum Size Solubilization of Membrane Proteins with Cocktails of Phospholipid Analogues.

Membrane proteins (MPs) play important roles in various cellular processes and are major targets for drugs. Solubilization of MPs is often needed for structural and biophysical studies. For high-resolution nuclear magnetic resonance measurements, there is a size limit of the sample (<100 kDa), and a high thermal stability at an increased temperature is required. Furthermore, lipid bilayer-like environments are desirable to preserve the native states of MPs. However, existing solubilization techniques do not fulfill these requirements at the same time. In this study, we combined two phospholipid analogues as a solubilizer and stabilizer to isolate MPs. This method maintained bacteriorhodopsin (bR) extracted from purple membranes in its native state for 7 d at 40 °C. The solubility was comparable to that of conventional detergents for MPs, and the thermal stability of the solubilizate was the best among them. The increase in the molecular size caused by the solubilization of bR was only 20 kDa, indicating that 20 phospholipid analogue molecules were sufficient to solubilize one bR molecule. ¹⁵N-¹H heteronuclear single quantum coherence spectra of solubilized ²H- and ¹⁵N-labeled bR gave ∼80% of the expected peaks. In addition, the lysate of human neuropeptide Y₂ receptor-expressing mammalian cells exhibited ligand recognition for 7 d at 37 °C, suggesting that this technique can be used for ligand screening. Moreover, the structure of the single membrane-spanning M2 protein of the influenza A virus expressed in Escherichia coli was stably maintained for 7 d at 40 °C. Thus, our method is promising for various MP studies.