Aqueous solubilization of transmembrane peptide sequences with retention of membrane insertion and function.
Aqueous solubilization of transmembrane peptide sequences with retention of membrane insertion and function.
复制标题
跨膜肽序列的水溶解并保留膜插入和功能。
DOI：
10.1016/s0006-3495(98)77784-7
复制
发表时间：
1998
影响因子：
3.4
通讯作者：
Iwamoto,T
中科院分区：
文献类型：
--
作者：
Tomich,JM;Wallace,D;Henderson,K;Mitchell,KE;Radke,G;Brandt,R;Ambler,CA;Scott,AJ;Grantham,J;Sullivan,L;Iwamoto,T
我们最近报道，当C-K4-M2GlyR被掺入培养的肾上皮细胞单层的顶膜时，它能模拟氯通道的作用。C-K4-M2GlyR是通过在大脑甘氨酸受体M2跨膜序列的N-端或C-端添加赖氨酸残基而制备的一系列多肽之一。这项研究解决了这些修饰如何影响物理性质，如水的溶解性、聚集性和二级结构，以及修饰的多肽在上皮单分子层中形成通道的能力。观察到M2GlyR跨膜序列在水介质中溶解度的逐步提高和聚集性的降低。部分但不是全部的多肽处理后，上皮单分子层的短路电流(ISC)增加。M2GlyR多肽越易溶、聚集越少，其生物活性越高。正如我们在之前的交流中所描述的，通道活性对氯离子通道阻滞剂二苯胺-2-羧酸盐和Na/K/2Cl共转运体的抑制剂布美他尼的敏感性被用来评估不同组装的通道形成肽的氯离子选择性的变化。未修饰的M2GlyR序列和正电荷较少的修饰多肽对这些药物比高电荷形式更敏感。这项研究表明，相对不溶的跨膜序列可以进行修饰，以便在没有有机溶剂的情况下更容易纯化和传递，并保留膜的结合、插入和组装。
We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (ISC) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.