Ion Channel Design Using Unnatural Amino Acids

近三分之一已完成的基因组序列和几乎一半可能成为药物设计目标的受体是完整的膜蛋白。因此，了解膜蛋白的结构和能量学是结构生物学的一个关键和未解决的问题。与可溶于水介质的蛋白质相比，对膜蛋白结构的稳定性和特异性的主要相互作用知之甚少。可溶性蛋白呈两部分结构，外部亲水，内部疏水。在膜蛋白中没有观察到这种二元模式，这使得设计成为一个难题。挑战在于如何在非极性环境中控制结构。本提案的长期目标是开发膜蛋白的设计元素。我们的方法依赖于高氟材料独特的相分离特性。拟议的研究将利用在非极性膜环境中由相分离驱动的折叠，适当定位的氟化侧链，以提供预定的结构和功能组合。本提案的具体目的是：(1)发展对映体纯氟化氨基酸类似物的合成方法；(2)利用生物物理技术探索含氟肽的三维结构；(3)通过选择性替换核心疏水残基，研究含氟相在蛋白质环境中稳定性的影响；(4)基于膜内相分离的跨膜成孔肽系设计与合成；(5)利用荧光、CD光谱、差示扫描和等温滴定量热相结合的方法对脂质囊泡和平面脂质膜中肽脂相互作用进行生物物理表征；(6)通过扫描力显微镜表征肽对脂质形态的影响并直接可视化孔隙形成；(7)利用单通道电导和荧光检测染料或H+释放来研究通道活性。最终，本文提出的设计和表征研究将有助于构建有效的膜转运剂，这将增加现有的抗生素，并有助于对抗细菌对当前治疗药物日益增加的耐药性。

Nearly a third of completed genomic sequences and almost half of all receptors that are likely to be targets for drug design are integral membrane proteins. Understanding the structure and energetics of membrane proteins is therefore a key and unsolved problem in structural biology. In contrast to proteins soluble in aqueous media, the primary interactions that contribute to the stability and specificity of membrane protein structures are poorly understood. Soluble proteins display a bipartite architecture with hydrophilic exteriors and hydrophobic interiors. This kind of binary patterning is not observed in membrane proteins, rendering design a difficult problem. The challenge is one of control over structure in nonpolar surroundings. The broad long term objective of this proposal is the development of design elements for membrane proteins. Our approach relies on the unique phase separation properties of highly fluorinated materials. The proposed studies will make use of folding driven by phase separation in the non polar membrane environment of appropriately positioned fluorinated side chains to deliver predetermined structural and functional ensembles. The specific aims of this proposal are: (1) To develop methodology for synthesis of enantiomerically pure fluorinated amino acid analogues; (2) To probe the three dimensional structure of fluorous peptides using biophysical techniques; (3) To study the effect of stability of fluorous phases in protein environments by selectively replacing core hydrophobic residues; (4) design and synthesis of membrane spanning and pore forming peptide ensembles based on phase separation within the membrane; (5) biophysical characterization of the peptide-lipid interactions in lipid vesicles and planar lipid membranes using a combination of fluorescence and CD spectroscopy, differential scanning and isothermal titration calorimetry; (6) characterization of the influence of peptides on lipid morphology and direct visualization of pore formation by scanning force microscopy and (7) investigation of channel activity using single channel conductance and fluorescence assays probing dye or H+ release. Ultimately the design and characterization studies proposed here should facilitate the construction of effective membrane transport agents that will add to currently available antibiotics and help in combating the ever- increasing resistance of bacteria to current therapeutic drugs.