Voltage-Gated Insertion of Colicin into Planar Bilayers

将大肠菌素电压门控插入平面双层

基本信息

批准号：
6850907
负责人：
William A. Cramer
金额：
$ 3.85万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-02-01 至 2007-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6850907
关键词：
Commonwealth of Independent States biophysics colicines conformation cooperative study fluorescence microscopy intermolecular interaction lipid bilayer membrane membrane proteins mutant pore forming protein protein structure function transposon /insertion element voltage /patch clamp voltage gated channel

项目摘要

DESCRIPTION (provided by applicant) Studies on membrane import and channel formation of the pore-forming colicins concern: the nature of (i) the large soluble --> membrane-bound structural transition undergone by colicins, toxins, and other membrane-active proteins, (ii) the surface-bound state that potentates helix insertion, (iii) structure changes associated with voltage-gated channel formation, and (iv) the pathway of protein insertion into the membrane, and (v) the mechanism by which the MW = 65,000 colicins are translocated across the E. coli outer membrane. The structure of the colicin E1 channel domain, solved at atomic resolution, allows structure-based mutagenesis strategies to test models for structural transitions upon membrane-binding and channel formation. Single-Trp and -Cys mutants were used in fluorescence quenching and fluorescence resonance energy transfer to define the colicin channel bound in the membrane interfacial layer as an extended, flexible, two-dimensional helical net. Planar lipid bilayer experiments have been carried out in collaboration with the lab of Y. N. Antonenko (Moscow, Russia) to observe the kinetics of colicin channel formation and related properties. There have been 4 collaborative projects: (1) Colicin channel activity was photoinactivated in the presence of sensitizing dyes, and this effect depended on the presence of Trp495 in helix 9 of channel domain. Colicin photoinactivation will serve as an important model for study of photodamage of membrane proteins and photodynamic therapy, widely used in cancer treatment. (2) Colicin import and channel formation was found to be very sensitive to membrane anionic lipid content and to be "tuned" at a surface potential of -60 +/- 5 mV. (3) The channel activities of purified outer membrane receptors proteins were found to be occluded by exogenous colicin. (4) Preliminary experiments indicate that colicin El membrane-binding and channel formation is affected by the lipid interfacial dipole potential. It is proposed to simultaneously measure channel current and fluorescence with horizontal planar bilayers and to analyze the kinetics and pathway of voltage-gated colicin insertion into, and channel formation in, the membrane.

描述（由申请人提供）对成孔大肠菌素的膜输入和通道形成的研究涉及：(i) 大肠菌素、毒素和其他膜活性蛋白经历的大可溶性→膜结合结构转变的性质，(ii) 表面增强螺旋插入的结合态，(iii) 与电压门控通道形成相关的结构变化，以及 (iv) 蛋白质插入膜的途径，以及 (v) MW = 65,000 大肠菌素的机制易位穿过大肠杆菌外膜。大肠菌素 E1 通道结构域的结构以原子分辨率解析，允许基于结构的诱变策略来测试膜结合和通道形成时的结构转变模型。单色氨酸和半胱氨酸突变体用于荧光猝灭和荧光共振能量转移，以将膜界面层中结合的大肠菌素通道定义为延伸的、柔性的二维螺旋网。与 Y.N.Antonenko 实验室（俄罗斯莫斯科）合作进行了平面脂质双层实验，以观察大肠菌素通道形成的动力学和相关特性。已有4个合作项目：（1）在敏化染料存在下，大肠菌素通道活性被光灭活，这种效应取决于通道结构域螺旋9中Trp495的存在。大肠杆菌素光失活将成为研究膜蛋白光损伤和光动力疗法的重要模型，广泛应用于癌症治疗。 (2) 发现大肠菌素输入和通道形成对膜阴离子脂质含量非常敏感，并且在-60 +/- 5 mV 的表面电位下进行“调节”。 (3)纯化的外膜受体蛋白的通道活性被外源大肠菌素封闭。 (4)初步实验表明，大肠菌素El膜结合和通道形成受脂质界面偶极电位的影响。建议使用水平平面双层同时测量通道电流和荧光，并分析电压门控大肠杆菌素插入膜中和通道形成的动力学和途径。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Chemical and photochemical modification of colicin E1 and gramicidin A in bilayer lipid membranes.

双层脂膜中大肠菌素 E1 和短杆菌肽 A 的化学和光化学修饰。

DOI：
10.1007/s00232-004-0674-y
发表时间：
2004
期刊：
The Journal of membrane biology
影响因子：
0
作者：
Sobko,AA;Vigasina,MA;Rokitskaya,TI;Kotova,EA;Zakharov,SD;Cramer,WA;Antonenko,YN
通讯作者：
Antonenko,YN

Tuning the membrane surface potential for efficient toxin import.

调节膜表面电位以有效导入毒素。

DOI：
10.1073/pnas.122613099
发表时间：
2002
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Zakharov,StanislavD;Rokitskaya,TatyanaI;Shapovalov,VladimirL;Antonenko,YuriN;Cramer,WilliamA
通讯作者：
Cramer,WilliamA

On the role of lipid in colicin pore formation.

关于脂质在大肠杆菌素孔形成中的作用。