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修饰和扩展。AlphaHL由金黄色葡萄球菌分泌，是一种水溶性的293个残基的多肽，其在脂质双层中形成七聚体孔。这是一个很好的目标，所提出的研究：三维结构是可用的，蛋白质是强大的，可以获得丰富的，它自组装成膜具有固定的亚基化学计量和在一个单一的方向，大量的突变体是可用的，蛋白质耐受极端的操纵结构，杂七聚体可以制备和纯化。此外，孔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，延长的开放期和弱离子选择性，alphaHL构成了蛋白质工程的空白。AlphaHL将通过环糊精、环肽、响应性聚合物、寡核苷酸和二分螯合剂的靶向非共价和共价修饰进行彻底重塑。所提出的操作，包括改变蛋白质腔的内部，以前没有在膜蛋白上进行过，并且很少在任何种类的蛋白质上进行。重要的是，我们不会满足于展示折叠和组装单独;我们的重点将是功能，特别是在单一电导，离子选择性和通道阻滞剂的敏感性的改变。预期成果是：（i）更好地理解跨膜通道的基本特性;（ii）创建具有自然界中未发现的新特性的孔;（iii）更好地理解从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、生物治疗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.