Lindhoud Simon, Carvalho Vanessa, Pronk Joachim W, Aubin-Tam Marie-Eve
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology . Lorentzweg 1, Delft 2628 CJ, The Netherlands.
Biomacromolecules. 2016 Apr 11;17(4):1516-22. doi: 10.1021/acs.biomac.6b00140. Epub 2016 Mar 22.
Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene-maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.
膜蛋白的纯化及其后续功能化过程中面临的挑战，常常使其生化和生物物理特性的表征变得复杂。膜蛋白的纯化通常涉及用去污剂分子取代蛋白质周围的脂质，这可能会影响蛋白质的结构和功能。最近有研究表明，苯乙烯 - 马来酸共聚物（SMA）能够将生物膜中的整合膜蛋白溶解到纳米尺寸的圆盘结构中。在这些纳米颗粒内，蛋白质嵌入其天然脂质双层的一个片段中，该片段通过像手镯一样包裹圆盘的两亲性聚合物在溶液中得以稳定。这种无去污剂纯化膜蛋白的方法有极大潜力简化纯化过程，但不利于将功能化合物与膜蛋白进行偶联。通常，这种功能化涉及费力地制备蛋白质变体以及优化标记程序，以确保对蛋白质的扰动最小。在此，我们提出一种策略，通过将半胱胺接枝到聚合物上来修饰SMA，从而规避其中的一些复杂问题。该反应生成具有溶剂暴露巯基（SMA-SH）的SMA，并允许对SH基团的覆盖度进行调节。尺寸排阻色谱、动态光散射和透射电子显微镜表明，SMA-SH能像SMA一样将脂质双层膜溶解成脂质纳米圆盘。此外，我们证明，与SMA一样，SMA-SH能将蛋白脂质体溶解成负载蛋白质的纳米圆盘。我们使用Alexa Fluor 488和生物素的硫醇反应性衍生物对SMA-SH-脂质纳米圆盘进行共价修饰。因此，SMA-SH有望同时应对膜蛋白纯化和功能化方面的挑战。
