Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, 60438 Frankfurt, Germany.
Institute of Physical and Theoretical Chemistry, Goethe University, 60438 Frankfurt, Germany.
Biochim Biophys Acta Biomembr. 2022 Nov 1;1864(11):184017. doi: 10.1016/j.bbamem.2022.184017. Epub 2022 Jul 31.
Nanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins. We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality.
由两亲性支架蛋白和小脂质双层组成的纳米颗粒是用于重建膜蛋白的功能和结构特性的有价值的工具。与无细胞蛋白质生产系统结合使用时，纳米颗粒可用于共翻译和独立转导插入膜蛋白到定制的脂质环境中。这种策略通过避免去污剂与新生膜蛋白接触，从而快速生成蛋白质/纳米颗粒复合物。经常使用的是用膜支架蛋白（MSP）或 Saposin A（SapA）支架的工程衍生物组装的纳米颗粒。此外，无细胞反应中存在几种形成膜蛋白/纳米颗粒复合物的策略。但是，尚不清楚这些策略如何影响无细胞合成的膜蛋白的功能折叠、寡聚体组装和膜插入效率。我们系统地研究了细胞游离合成的菌视紫红质（PR）的插入效率和样品质量，该 PR 是在 MSP 和 SapA 基纳米颗粒中进行共翻译插入的。分析了三种可能的 PR/纳米颗粒形成策略：（i）PR 整合到供应的预组装纳米颗粒中，（ii）在 PR 表达时从供应的支架蛋白和脂质共组装纳米颗粒，以及（iii）在供应的脂质存在下共同表达支架蛋白和 PR。分析了来自三种策略生成的样品的产率、均一性以及 PR 高寡聚复合物的形成。为 PR 找到的最佳条件应用于 G 蛋白偶联受体的合成。该研究为通过不同过程快速合成膜蛋白/纳米颗粒样品提供了全面的指南，并确定了调节样品产率和质量的关键参数。
