Morrison Emma A, Henzler-Wildman Katherine A
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St.Louis, MO, USA.
Biochim Biophys Acta. 2012 Mar;1818(3):814-20. doi: 10.1016/j.bbamem.2011.12.020. Epub 2011 Dec 29.
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.
将整合膜蛋白重构到适合进行生物物理和结构研究的膜模拟环境中一直是一项挑战。各向同性双分子层囊泡兼具两者优势——使膜蛋白被一小片形成双层的脂质包围，同时又足够小，能够各向同性地翻滚并产生良好的溶液核磁共振谱。然而，将膜蛋白重构到各向同性双分子层囊泡中的传统方法会使蛋白暴露于潜在的不稳定环境中。先将蛋白重构到脂质体中，然后向该混合物中添加短链脂质，可产生双分子层囊泡样品，同时将蛋白暴露于不利环境的程度降至最低。结果是重构到双分子层囊泡中的蛋白产量更高，长期稳定性、均一性和样品间的可重复性得到改善。这表明在重构过程中蛋白结构得到了更好的保留，从而降低了每个样品的成本，减少了样品的制备数量，并缩短了采集高质量谱所需的核磁共振时间。此外，这种方法能够将蛋白重构到具有更广泛脂质组成的各向同性双分子层囊泡中。小多药耐药转运蛋白EmrE（一种已知对其环境高度敏感的蛋白）的实验证明了这些结果。
