The functional role of Cys3–Cys4 loop in hydrophobin HGFI
The functional role of Cys3–Cys4 loop in hydrophobin HGFI
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DOI：
10.1007/s00726-014-1805-0
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发表时间：
2014-09
期刊：
影响因子：
3.5
通讯作者：
Baolong Niu;Yanbo Gong;Xianghua Gao;Haijin Xu;M. Qiao;Wenfeng Li
中科院分区：
文献类型：
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作者：
Baolong Niu;Yanbo Gong;Xianghua Gao;Haijin Xu;M. Qiao;Wenfeng Li
疏水蛋白是一大类低分子量蛋白质。这些蛋白质具有高度的表面活性，可以通过在疏水-亲水界面自组装形成两亲性膜。根据疏水蛋白的物理性质和亲水性，疏水蛋白可分为两类。在氨基酸序列和高级结构分析的基础上，一些模型表明，Cys 3-Cys 4环区在I类和II类疏水蛋白中可以表现出显着的差异，在它们的排列和构象，并在棒状结构的形成中具有关键作用。为了研究I类疏水蛋白中Cys 3-Cys 4环的需要，我们使用蛋白融合技术，通过将来自灰树花的I类疏水蛋白HGFI的Cys 3和Cys 4之间的氨基酸替换为来自里氏木霉的II类疏水蛋白HFBI的Cys 3和Cys 4之间的氨基酸，获得突变蛋白HGFI-AR。突变蛋白HGFI-AR基因在毕赤酵母中获得了高效表达。水接触角（WCA）和X射线光电子能谱（XPS）测试表明，纯化的HGFI-AR可在云母和疏水聚苯乙烯表面自组装形成两亲性膜。这种特性使他们能够改变聚苯乙烯和云母的表面润湿性，并改变硅化玻璃的元素组成。与重组I类疏水蛋白HGFI（rHGFI）相比，通过HGFI-AR在疏水表面上形成的膜不足以抵抗1%热SDS洗涤。原子力显微镜（AFM）测量表明，与rHGFI不同，在突变蛋白HGFI-AR涂覆的云母表面上没有观察到小棒结构。此外，与rHGFI相比，HGFI-AR在水-空气界面自组装后，圆二色性（CD）光谱未检测到二级结构变化。HGFI-AR不能被认为是硫磺素T（THT）的荧光强度增加和刚果红（CR）吸收光谱偏移（在THT（CR）/HGFI-AR混合水溶液剧烈涡旋后）的原因。值得注意的是，Cys 3-Cys 4环的替换可损害I类疏水蛋白HGFI的小棒形成。因此，可以推测，当I类疏水蛋白HGFI在疏水-亲水界面自组装时，Cys 3-Cys 4环在构象和功能上起重要作用。
Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic–hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3–Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3–Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI fromGrifola frondosawith those ones between Cys3 and Cys4 of the class II hydrophobin HFBI fromTrichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed inPichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water–air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3–Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3–Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic–hydrophilic interfaces.