Henrich Erik, Peetz Oliver, Hein Christopher, Laguerre Aisha, Hoffmann Beate, Hoffmann Jan, Dötsch Volker, Bernhard Frank, Morgner Nina
Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J W Goethe-University, Frankfurt am Main, Germany.
Institute of Physical and Theoretical Chemistry, J W Goethe-University, Frankfurt am Main, Germany.
Elife. 2017 Jan 9;6:e20954. doi: 10.7554/eLife.20954.
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
膜蛋白在其自然脂质环境中经常组装成更高阶的同型或异型寡聚体。这种复合物的形成可以调节它们的折叠、活性以及底物选择性。因此，避免关键步骤（如膜崩解、转移到人工环境或化学修饰）的非破坏性方法对于分析天然膜蛋白组装的分子机制至关重要。无细胞合成生物学、纳米盘技术和非共价质谱的结合为分析限定膜内的膜蛋白寡聚化提供了出色的协同作用。我们通过对各种膜蛋白的寡聚状态进行表征来举例说明我们的策略，这些膜蛋白包括离子通道、转运蛋白和组装成六聚体复合物的膜整合酶。我们进一步指出，MraY转位酶的脂质依赖性二聚体形成与酶活性相关。膜蛋白/纳米盘样品的无去污剂合成以及通过LILBID质谱进行的分析为在天然环境中分析膜蛋白提供了一个通用平台。
