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显著改变和扩展。AlphaHL由金黄色葡萄球菌分泌，是一种水溶性的293残基多肽，在脂质双分子层中形成七聚体孔。它是一个极好的研究目标：三维结构是可用的，蛋白质是健壮的，可以大量获得，它自组装成具有固定亚基化学计量和单一取向的膜，大量的突变体是可用的，蛋白质在结构上耐受极端的操作，可以制备和纯化异七聚体。此外，孔隙的功能特性可以通过单通道记录进行复杂的详细检查。由于其高电导率、长开放期和弱离子选择性，alphaHL成为蛋白质工程的一块空白。AlphaHL将通过环糊精、环肽、反应性聚合物、寡核苷酸和两部分螯合剂进行靶向非共价和共价修饰，从根本上进行重塑。所提出的操作，包括改变蛋白质腔的内部，以前还没有在膜蛋白上进行过，而且很少在任何一类蛋白质上进行过。重要的是，我们不会满足于仅仅展示折叠和组装；我们的重点将放在功能上，特别是在单一电导、离子选择性和通道阻滞剂敏感性方面的变化。预期结果是：(i)更好地理解跨膜通道的基本特性；（ii）产生具有自然界中没有的新性质的孔隙；（iii）更好地理解由通常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，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.