Huang Yen-Hua, Colgrave Michelle L, Daly Norelle L, Keleshian Asbed, Martinac Boris, Craik David J
Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Australia.
J Biol Chem. 2009 Jul 31;284(31):20699-707. doi: 10.1074/jbc.M109.003384. Epub 2009 Jun 1.
The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defense-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B1, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B1 showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata B1 was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 41-47 A. Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides.
环肽是一类包含胱氨酸结基序的大型环状微型蛋白质。它们在植物中作为与防御相关的蛋白质表达，具有杀虫活性。在这里，我们研究它们在膜相互作用和破坏中的作用。发现原型环肽卡拉塔B1可诱导自猝灭荧光团羧基荧光素从磷脂囊泡中泄漏。对卡拉塔B1进行丙氨酸扫描诱变表明，裂解活性所必需的残基聚集在一起，形成一个生物活性面。卡拉塔B1被隔离在膜表面，并且从囊泡中解离缓慢。电生理实验表明，在与卡拉塔B1孵育时，脂质体膜片上会诱导形成导电孔。根据电流-电压关系计算出的电导率表明，在双层膜片中形成的孔的直径为41-47埃。总的来说，这些发现为赋予这个迷人的超稳定大环肽家族的生物功能多样性提供了一个机制解释。
