Department of Biochemistry, University of Illinois, Urbana, IL, 16801, USA.
Department of Biochemistry, University of Illinois, Urbana, IL, 16801, USA; Department of Chemistry, University of Illinois, Urbana, IL, 16801, USA.
Anal Biochem. 2020 Oct 15;607:113860. doi: 10.1016/j.ab.2020.113860. Epub 2020 Aug 1.
We describe the construction, expression and purification of three new membrane scaffold proteins (MSP) for use in assembling Nanodiscs. These new MSPs have a variety of luminescent properties for use in combination with several analytical methods. "Dark" MSP has no tryptophan residues, "Ultra-Dark" replaces both tryptophan and tyrosine with non-fluorescent side chains, and "Ultra-Bright" adds additional tryptophans to the parent membrane scaffold protein to provide a dramatic increase in native tryptophan fluorescence. All MSPs were used to successfully assemble Nanodiscs nominally 10 nm in diameter, and the resultant bilayer structure was characterized. An example of the usefulness of these new scaffold proteins is provided.
我们描述了三种新的膜支架蛋白（MSP）的构建、表达和纯化，用于组装纳米盘。这些新的 MSP 具有多种发光特性，可与多种分析方法结合使用。“暗”MSP 没有色氨酸残基，“超暗”用非荧光侧链取代色氨酸和酪氨酸，“超亮”在母体膜支架蛋白上添加额外的色氨酸，以提供天然色氨酸荧光的显著增加。所有 MSP 都成功地组装了直径约为 10nm 的纳米盘，并且对所得双层结构进行了表征。提供了这些新支架蛋白的一个应用实例。
