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De novo synthesis of a small globular protein SGP and its insertion into lipid bilay

小球状蛋白SGP的从头合成及其插入脂质双层

基本信息

批准号：
07680729
负责人：
LEE Sannamu
金额：
$ 1.47万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

The question of how to design a water-soluble globular protein remains. We report here a making of a native-like and pore-forming small globular protein (SGP,69 amino acid residues). The protein was designed to have four helices : a Trp-containing short hydrophobic helix in a middle surrounded by three Tyr-containing long basic amphiphilic helices. Size exclusion chromatography and CD measurement indicated that in buffer solution SGP is monomeric with a 50% helical structure. SGP did not completely denature even at high temperature (90ﾟC) ard at relatively high Gu-Cl concentration that the denaturant concentration at the midpoint of transition is 5M.Dye-binding studies and fluorescence energy transfer experiments showed that SGP possesses a hydrophobic binding site and its Trp of central helix is present at relatively hydrophobic region and accepts the energy from Tyr (s) in other amphiphilic helices, indicating that SGP takes a stable globular-like structure in aqueous solution. From the depth dependent fluorescent studies using egg PC liposomes containing n-doxyl fatty acids and brominated phospholipid as quenchers, it was found that the hydrophobic central alpha-helix is able to enter spontaneously into the lipid bilayrs and the Trp in central alpha-helix is located at about the middle of the alkyl chain in the outer layr of the phospholipid bilayr. The peptide is also able to increase the membrane permeability with two modes of current (basal current and single ion channel) in planar phospholipid bilayrs, indicating that the spontaneous insertion of the protein into lipid bilayr (basal current) and then the formation of a uniform size of channel pore (14pS). SGP is useful as a basic and starting model to find good amino acid sequences that fold to a desired protein structure and to search translocation mechanisms from aqueous solution into lipid bilayrs.

如何设计一种水溶性球形蛋白的问题仍然存在。我们在这里报告了一个天然的和形成孔的小球状蛋白（SGP，69个氨基酸残基）的制造。该蛋白被设计成有四个螺旋：中间是一个含有trp的短疏水螺旋，周围是三个含有tyrr的长基本两亲螺旋。不相容色谱和CD测定表明，缓冲溶液中SGP为单体，螺旋结构为50%。即使在高温（90℃）和较高的cu - cl浓度（过渡中点变性剂浓度为5M）下，SGP也没有完全变性。染料结合研究和荧光能量转移实验表明，SGP具有疏水结合位点，其中心螺旋的Trp存在于相对疏水区域，并接受来自其他两亲螺旋上Tyr (s)的能量，表明SGP在水溶液中呈稳定的球状结构。以含n-羟脂肪酸和溴化磷脂的蛋PC脂体为猝灭剂进行深度依赖荧光研究，发现疏水中心α -螺旋能够自发进入脂质双分子层，中心α -螺旋上的Trp位于磷脂双分子层外层烷基链的中间左右。该肽还能在平面磷脂双层膜中以两种电流模式（基流和单离子通道）增加膜通透性，表明该蛋白自发插入脂双层膜（基流），然后形成均匀大小的通道孔（14pS）。SGP是一个基本的和开始的模型，用于寻找折叠成所需蛋白质结构的良好氨基酸序列，以及搜索从水溶液到脂质双层的易位机制。