Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and decoration proteins in the development of robust nanoscale display vehicles
Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and decoration proteins in the development of robust nanoscale display vehicles
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DOI：
10.1016/j.biomaterials.2012.04.026
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发表时间：
2012-08-01
期刊：
影响因子：
14
通讯作者：
Teschke, Carolyn M.
中科院分区：
文献类型：
--
作者：
Parent, Kristin N.;Deedas, Christina T.;Teschke, Carolyn M.
建立了利用噬菌体P22衣壳和噬菌体装饰蛋白(DEC)展示货物的逐步添加方法。结合了纳米金标记的DEC的P22颗粒冷冻水化样品的三维图像重建揭示了DEC的N-末端和C-末端的位置。每个末端都很容易通过镍-氮三乙酸等亲和标记进行分子展示，总共提供了240个货物结合位点。圆二色谱表明DEC是一种富含β-折叠的蛋白质，荧光各向异性结合实验表明DEC与P22头部具有很高的亲和力(约110 nM)。DEC还与P22纳米管结合，P22纳米管是当P22外壳蛋白包含F170A氨基酸取代时形成的螺旋对称组件。用冷冻电子显微镜观察了几类与DEC结合的管子，并用螺旋重建的方法确定了它们的三维结构。在所有情况下，DEC三聚体与P22衣壳和纳米管结合的位置是三个相邻的胶囊(六个外壳蛋白亚基的寡聚体)彼此紧密相连的位置。Dec和P22之间稳定的相互作用使强大的纳米级展示车辆的发展成为可能。(C)2012爱思唯尔有限公司。保留所有权利。
A stepwise addition protocol was developed to display cargo using bacteriophage P22 capsids and the phage decoration (Dec) protein. Three-dimensional image reconstructions of frozen-hydrated samples of P22 particles with nanogold-labeled Dec bound to them revealed the locations of the N- and C-termini of Dec. Each terminus is readily accessible for molecular display through affinity tags such as nickel-nitrilotriacetic acid, providing a total of 240 cargo-binding sites. Dec was shown by circular dichroism to be a beta-sheet rich protein, and fluorescence anisotropy binding experiments demonstrated that Dec binds to P22 heads with high (similar to 110 nM) affinity. Dec also binds to P22 nanotubes, which are helically symmetric assemblies that form when the P22 coat protein contains the F170A amino acid substitution. Several classes of tubes with Dec bound to them were visualized by cryo-electron microscopy and their three-dimensional structures were determined by helical reconstruction methods. In all instances, Dec trimers bound to P22 capsids and nanotubes at positions where three neighboring capsomers (oligomers of six coat protein subunits) lie in close proximity to one another. Stable interactions between Dec and P22 allow for the development of robust, nanoscale size, display vehicles. (C) 2012 Elsevier Ltd. All rights reserved.