MEMBRANE PROTEIN ENGINEERING BY TARGETED MODIFICATION

葡萄球菌α-溶血素(AlphaHL)的靶向修饰将带来跨膜孔功能特性的戏剧性修改和扩展。AlphaHL是由金黄色葡萄球菌分泌的一种水溶性多肽，由293个残基组成，在脂双层中形成七聚体孔。对于建议的研究来说，它是一个很好的目标：三维结构可用，蛋白质是健壮的，可以得到丰富的，它以固定的亚单位化学计量比自组装成膜，并且在一个单一的取向上，可以获得大量的突变体，蛋白质在结构上可以忍受极端的操作，可以制备和纯化异七聚体。此外，可以通过单通道记录来复杂地检查孔的功能属性。由于其高电导、长开放阶段和弱离子选择性，αHL构成了蛋白质工程的一张白板。AlphaHL将通过环糊精、环肽、响应性聚合物、寡核苷酸和两部分螯合剂的靶向非共价和共价修饰进行根本性改造。所提出的操作，包括改变蛋白质腔的内部，以前从未在膜蛋白质上进行过，也很少对任何一类蛋白质进行过。重要的是，我们不会满足于仅展示折叠和组装；我们的重点将放在功能上，特别是在单位电导、离子选择性和对通道阻滞剂的敏感性方面的变化。预期的结果是：(1)更好地了解跨膜通道的基本性质；(2)创造具有自然界中没有的新性质的孔；(3)更好地理解由正常情况下不相互作用的生物大分子构建模块化纳米结构的规则。这项工作将通过为生物传感器、细胞保存、生物疗法和药物输送提供新技术来影响医疗保健。

Dramatic modifications and extensions of the functional properties of a transmembrane pore will be brought about by targeted modification of staphylococcal alpha-hemolysin (alphaHL). AlphaHL is secreted by Staphylococcus aureus is a water-soluble, 293-residue polypeptide, which forms heptameric pores in lipid bilayers. It is an excellent target for the proposed studies: three dimensional structures are available, the protein is robust and can be obtained in abundance, it self-assembles into membranes with a fixed subunit stoichiometry and in a single orientation, a large collection of mutants is available, the protein tolerates extreme manipulations in structure, heteroheptamers can be prepared and purified. Further, the functional properties of the pore can be examined in intricate detail by single channel recording. Given its high conductance, prolonged open stage and weak ion selectivity, alphaHL constitutes a blank slate for protein engineering. AlphaHL will be radically remodeled by targeted non-covalent and covalent modifications with cyclodextrins, cyclic peptides, responsive polymers, oligonucleotides and a bipartite chelator. The manipulations proposed, including the alteration of the inside of a protein cavity, have not been carried out previously on membrane proteins and have been performed only rarely on any class of protein. Importantly, we will not be content to demonstrate folding and assembly alone; our focus will be on function, especially alterations in unitary conductance, ion selectivity, and susceptibility to channel blockers. Expected outcomes are: (i) a better understanding of the fundamental properties of transmembrane channels; (ii) the creation of pores with new properties not found in nature; (iii) a better understanding of the rules governing the construction of modular nanostructures from biological macromolecules that do not normally interact. The work will impact healthcare, by providing new technology for biosensors, cell preservation, biotherapeutics and drug delivery.