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

如何设计一种可溶于水的球状蛋白的问题仍然存在。我们在这里报道了一种天然的、形成孔的小球状蛋白(SGP，69个氨基酸残基)的制备。该蛋白质被设计成有四个螺旋：中间有一个含有Trp的短疏水螺旋，周围环绕着三个含有Tyr的长的碱性两亲性螺旋。大小排斥层析和圆二色谱分析表明，在缓冲溶液中，SGP为单体，具有50%的螺旋结构。SGP即使在高温(90ﾟC)和较高的Gu-Cl浓度下也没有完全变性，转变中点的变性剂浓度为5M。染料结合和荧光能量转移实验表明，SGP具有疏水结合部位，其中心螺旋的色氨酸存在于相对疏水的区域，并接受来自TYR(S)的能量，表明SGP在水溶液中呈稳定的球状结构。用含N-羟基脂肪酸和溴化磷脂的鸡蛋PC脂质体作为猝灭剂进行的深度依赖荧光研究发现，疏水的中心α-螺旋能够自发地进入脂双层，中心α-螺旋中的色氨酸位于磷脂双层外层的烷基链的中间。该多肽还能以两种方式(基础电流和单离子通道)增加磷脂双层膜的通透性，表明蛋白质自发地插入到脂质双分子层(基础电流)，进而形成大小均匀的通道孔(14ps)。SGP是寻找折叠成所需蛋白质结构的良好氨基酸序列和探索从水溶液到脂质双分子层的转运机制的一个基本和开始的模型。

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.