Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile

Reconstitution of integral membrane proteins into membrane mimetic environments suitable for biophysical and structural studies has long been a challenge. Isotropic bicelles promise the best of both worlds-keeping a membrane protein surrounded by a small patch of bilayer-forming lipids while remaining small enough to tumble isotropically and yield good solution NMR spectra. However, traditional methods for the reconstitution of membrane proteins into isotropic bicelles expose the proteins to potentially destabilizing environments. Reconstituting the protein into liposomes and then adding short-chain lipid to this mixture produces bicelle samples while minimizing protein exposure to unfavorable environments. The result is higher yield of protein reconstituted into bicelles and improved long-term stability, homogeneity, and sample-to-sample reproducibility. This suggests better preservation of protein structure during the reconstitution procedure and leads to decreased cost per sample, production of fewer samples, and reduction of the NMR time needed to collect a high quality spectrum. Furthermore, this approach enabled reconstitution of protein into isotropic bicelles with a wider range of lipid compositions. These results are demonstrated with the small multidrug resistance transporter EmrE, a protein known to be highly sensitive to its environment.