A fluorescent hinge peptide for the dimerization of proteins through disulfide formation

我们最近描述了一种二硫键桥联二聚肽，它是通过12个氨基酸基序CX3CX2CX3C(C=半胱氨酸，X=其他氨基酸)的氧化折叠形成的。半胱氨酸的含量很高--每三个氨基酸中就有半胱氨酸--超过了天然半胱氨酸基序，并在空气氧化时产生了独特的同质二聚选择性。分离的多肽不仅经过共价二聚，而且还通过重组DNA技术将大小为150 kDa的蛋白质融合到多肽上。其中一个同源二聚体多肽显示蓝色荧光，这可能是由其两个色氨酸之间的激基异构体形成的。我们将研究这种效应的结构要求，以增加这种铰链二聚体作为荧光标记的应用潜力。二聚化过程和氧化折叠的分子需求将通过系统的结构修改来研究。我们将尝试通过互补的合成和分析策略来确定八种半胱氨酸氧化折叠平衡行为的中间体。二聚化过程的结构要求将通过半胱氨酸、转位氨基酸和侧链电荷的系统氨基酸变化来测试，特别是异二聚肽。光谱方法将被用来表征铰链的两个半部分的构象迁移率，从它对结构环境的系统变化的响应，即用不同大小的多肽来“加载”铰链。这项应用的中心目的是确定四二硫化物二聚体多肽的结构先决条件，因为它对不同的蛋白质负荷具有高度的耐受性，并且具有独特的氧化折叠区域选择性。对这种新的蛋白质二聚基序的物理化学性质、构象和合作迁移率的研究将对多肽/蛋白质的折叠和稳定性具有普遍的意义。

We recently described a disulfide-bridged dimeric peptide which forms through oxidative folding of the 12 amino acids motif CX3CX2CX3C (C = cysteine, X = other amino acids). The high cysteine content – every third amino acid is cysteine – exceeds that of natural cysteine motifs and results in a unique homodimerization selectivity upon air oxidation. Not only the isolated peptide undergoes covalent dimerization but also proteins up to a size of 150 kDa that are fused to the peptide by recombinant DNA technology. One of the homodimeric peptide shows blue fluorescence that is probably caused by the excimer formation between its two tryptophans. We will investigate the structural requirements of this effect that increases the application potential of this hinge dimers as fluorescence tags. The molecular requirements of the dimerization process and the oxidative folding will be studied by systematic structural modifications. We will try to identify intermediates of the balance act of oxidative folding of eight cysteines by complementary synthetic and analytic strategies. The structural requirements of the dimerization process will be tested by systematic amino acid variations at the cysteines, the turn amino acids, and the side chain charges with a special focus on the heterodimeric peptides. Spectroscopic methods will be used to characterize the conformational mobility of the two halves of the hinge from its response to systematic variations of the structural environment i.e. the “loading” of the hinge with polypeptides of different size. The central aim of this application is to identify the structural prerequisites of the tetradisulfide dimeric peptide for its high tolerance to different protein loads and its unique regioselectivity of oxidative folding. The investigation of physicochemical properties, conformation, and cooperative mobility of this novel protein dimerization motif will be of interest for peptide/protein folding and stability in general.