Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Department of Physics, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Cell Chem Biol. 2020 Feb 20;27(2):245-251.e3. doi: 10.1016/j.chembiol.2019.11.008. Epub 2019 Dec 9.
Biological membranes are complex barriers in which membrane proteins and thousands of lipidic species participate in structural and functional interactions. Developing a strategic approach that allows uniform labeling of membrane proteins while maintaining a lipidic environment that retains functional interactions is highly desirable for in vitro fluorescence studies. Herein, we focus on complementing current methods by integrating the powerful processes of unnatural amino acid mutagenesis, bioorthogonal labeling, and the detergent-free membrane protein solubilization based on the amphiphilic styrene-maleic acid (SMA) polymer. Importantly, the SMA polymer preserves a thermodynamically stable shell of phospholipids. The approach that we present is both rapid and generalizable providing a population of uniquely labeled membrane proteins in lipid nanoparticles for quantitative fluorescence-based studies.
生物膜是复杂的屏障，其中膜蛋白和数千种脂质参与结构和功能相互作用。开发一种策略性的方法，允许均匀标记膜蛋白，同时保持保留功能相互作用的脂质环境，这对于体外荧光研究是非常理想的。在这里，我们通过整合非天然氨基酸诱变、生物正交标记以及基于两亲性苯乙烯-马来酸（SMA）聚合物的无去污剂膜蛋白溶解等强大过程，来补充当前的方法。重要的是，SMA 聚合物保留了热力学稳定的磷脂壳。我们提出的方法快速且具有通用性，可在脂质纳米颗粒中提供独特标记的膜蛋白群体，用于基于定量荧光的研究。
