DNA-corralled nanodiscs for study of large membrane proteins and their complexes

项目摘要 磷脂纳米盘在过去的二十年里吸引了人们的极大兴趣，作为一种提供一种原生的- 如双层环境，用于包埋膜蛋白的研究。一个流行的版本是由两个 来自载脂蛋白A1的膜支架蛋白(MSPs)的拷贝，形成双层带 在直径约10 nm的脂片疏水周长周围。我们最近报道了重组 MSP的不同版本，在与索尔特酶环化后，能够组装直径达80 nm的纳米盘。 然而，最大的那些容易融合和聚集。在这里，我们建议构建DNA-折纸 将多个10 nm MSP纳米盘重组成从60 nm到1000 nm的更大纳米盘的畜栏 在直径上。DNA畜栏还可以作为防止不必要的聚集的保险箱，并可以使 通过系在畜栏上，控制插入的客人的化学计量、几何形状和方向。我们会 研究中型(直径60 nm)纳米盘簇，每个具有不同的倾斜角，以及大型 用于捕获高密度客膜蛋白的(&gt；200 nm直径)纳米盘 或晶体排列，用于低温电子显微镜分析。一个主要好处将是通过中介实现更快的数据收集 通过呈现高密度的嘉宾。以晶体形式呈现可能会减少构象 可变性，这可以导致改进的颗粒分类。这项建议的主要目标如下 在目标1中，我们将生产直径从60 nm到1μm的dna-corrated Nanopes(DCND)，用于 以高密度承载脂质嵌入的膜蛋白。我们将调查DNA-折纸“桶”与外部 允许横向聚集的修改，允许客户MSP纳米盘自由旋转的内部修改， 以及能够组装双层MSP纳米盘的内部修饰。我们将调查DNA-折纸 “Arenas”，由刚性的V形楔形的自限均聚物组成，它将MSP纳米盘包裹在 直径从200 nm到1000 nm。在目标2中，我们将优化协议以实现大容量数据的可靠嵌入 DNA纳米盘中的膜蛋白及其复合体。特别是，我们将设计DNA- 重组SMA纳米盘提取天然线粒体VDAC/ANT1/HK复合体的研究 膜。我们还将设计不对称的、DNA排列的纳米盘来研究脂质分布。 位于同一面上的不同亚型的翻转酶。在目标3中，我们将使用冷冻EM进行结构分析 在DCNds中承载膜蛋白及其复合体。(I)我们将VDAC-1的复合体与 DCNds中己糖激酶酶、肌酸激酶和ANT1及其结构的电子显微镜研究 技巧。(Ii)我们将在含有不对称双层的DCND中放置CCR5和CD4的多个副本 匹配免疫细胞膜以研究与HIV1 gp160或更小构建体的相互作用。(Iii)我们 将使用装饰有辅助受体的相同DCND来成像与非传染性HIV-1病毒的相互作用- 如粒子(VLP)。负染和低温电子显微镜将被用来对感兴趣的系统进行成像。

Project Summary Phospholipid nanodiscs have attracted great interest over the last two decades as a means to provide a native- like bilayer environment for study of embedded membrane proteins. A popular version is assembled with two copies of membrane-scaffold proteins (MSPs), derived from Apolipoprotein A1, that form a double-layer belt around the hydrophobic perimeter of a lipid patch about 10 nm in diameter. We recently reported recombinant versions of MSP that, after cyclization with sortase, enable assembly of nanodiscs up to 80 nm in diameter. However, the largest ones are prone to fusion and aggregation. Here we propose to construct DNA-origami corrals that direct the reconstitution of multiple 10 nm MSP nanodiscs into larger ones from 60 nm to 1000 nm in diameter. DNA corrals additionally act as bumper cases to prevent unwanted aggregation and can enable control over stoichiometry, geometry, and orientation of inserted guests through tethering to the corral. We will investigate clusters of medium-sized (60 nm diameter) nanodiscs, each with a varying tilt angle, and large-sized (>200 nm diameter) nanodiscs for capture for a high density of guest membrane proteins, in either noncrystalline or crystalline arrangement, for cryoEM analysis. A major benefit will be in mediating faster data collection through presentation of high-density of guests. Presentation in crystalline format could reduce conformational variability, which can lead to improved particle classification. The primary objectives of this proposal are as follows: In Aim 1, we will generate DNA-corralled nanodiscs (DCNDs) from 60 nm up to 1 μm in diameter for hosting lipid- embedded membrane proteins at high density. We will investigate DNA-origami “barrels” with outer modifications that enable lateral clustering, inner modifications that enable free rotation of guest MSP nanodiscs, and inner modifications that enable assembly of double-decker MSP nanodiscs. We will investigate DNA-origami “arenas”, composed as self-limiting homopolymers of rigid V-shaped wedges, that enclose MSP nanodiscs with a diameter from 200 nm to 1000 nm. In Aim 2, we will optimize protocols for reliable embedding of large membrane proteins and their complexes in DNA-corralled nanodiscs. In particular, we will engineer DNA- corralled SMA nanodiscs and use them to extract VDAC/ANT1/HK complex from native mitochondrial membranes. We also will engineer asymmetric, DNA-corralled nanodiscs with respect to lipid distribution using different subtypes of flippases positioned on one face. In Aim 3, we will use cryoEM for structural analysis of hosted membrane proteins and their complexes in DCNDs. (i) We will embed complexes of VDAC-1 with hexokinases, creatine kinases and ANT1 in DCNDs and study their structures with electron microscopy techniques. (ii) We will place multiple copies of CCR5 and CD4 in DCNDs containing asymmetric bilayers matching the immune cell membrane to study the interaction with the HIV1 gp160 or smaller constructs. (iii) We will use the same DCND decorated with co-receptors to image the interaction with non-infectious HIV-1 virus- like particles (VLPs). Negative stain and cryoEM will be used to image the systems of interest.