Liu Shixuan, Li Shuang, Yang Yihu, Li Weikai
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Sci Adv. 2020 Dec 18;6(51). doi: 10.1126/sciadv.abe3717. Print 2020 Dec.
Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.
小膜蛋白是结构表征的困难靶点。在此，我们通过用可缔合的蛋白质实体限制其氨基和羧基末端来稳定其折叠，以超级折叠绿色荧光蛋白的两个半部分为例。末端受限的蛋白质具有功能，并且在过表达和纯化过程中显示出更高的稳定性。重新组装的绿色荧光蛋白为膜蛋白结晶提供了一个通用支架，能够衍射到原子分辨率，并有助于晶体识别、相位确定和密度修正。这种策略产生了来自不同功能家族的五种脊椎动物蛋白的14个新结构，极大地扩展了小膜蛋白的结构数据库。此外，细菌二硫键形成蛋白B（DsbB）的高分辨率结构表明，这种硫醇氧化还原酶通过一个催化三联体被激活，类似于半胱氨酸蛋白酶。总体而言，末端限制被证明对于小膜蛋白的稳定和结构测定非常有效。
