Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Sci Rep. 2013;3:2556. doi: 10.1038/srep02556.
Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochrome-P4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.
虽然高分辨率结构和膜蛋白动力学的重要性已得到广泛认可，但优化样品条件以保持膜蛋白的天然折叠和功能，使其适用于核磁共振（NMR）或 X 射线测量一直是一个主要挑战。虽然双分子层已被证明可以稳定膜蛋白的功能，并且越来越多地被用作模型膜，但它们在低温下失去磁定向，使得它们不适合研究热敏感的膜蛋白，如细胞色素 P450 和蛋白质-蛋白质复合物。在这项研究中，我们报告了具有温度抗性的双分子层，它们可以在很宽的温度范围内进行磁定向，并通过固态 NMR 光谱证明了它们在全长微粒体细胞色素 P450 和细胞色素 b5 的结构研究中的优势。我们的结果表明，兔细胞色素 P4502B4 的 N 端区域通常被切除以获得晶体结构，该区域是螺旋的，并且与脂质双层法线的倾斜角度约为 17°。
