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接的二聚体肽，其通过12个氨基酸基序CX 3CX 2CX 3C（C =半胱氨酸，X =其他氨基酸）的氧化折叠形成。高半胱氨酸含量-每三个氨基酸中有一个是半胱氨酸-超过了天然半胱氨酸基序的含量，并在空气氧化时产生独特的同源二聚选择性。不仅分离的肽经历共价二聚化，而且通过重组DNA技术与肽融合的高达150 kDa大小的蛋白质也经历共价二聚化。其中一个同源二聚体肽显示蓝色荧光，这可能是由它的两个双链体之间形成的激基缔合物引起的。我们将研究这种效应的结构要求，增加这种铰链二聚体作为荧光标记的应用潜力。将通过系统的结构修饰来研究二聚化过程和氧化折叠的分子要求。我们将尝试通过互补的合成和分析策略来鉴定八个半胱氨酸氧化折叠平衡作用的中间体。二聚化过程的结构要求将通过在半胱氨酸、转角氨基酸和侧链电荷处的系统性氨基酸变化进行测试，特别关注异二聚体肽。将使用光谱方法来表征铰链的两半的构象移动性，其来自其对结构环境的系统变化的响应，即铰链与不同大小的多肽的“负载”。本申请的中心目的是鉴定四二硫化物二聚体肽的结构先决条件，其对不同蛋白质负载的高耐受性和其独特的氧化折叠区域选择性。这种新的蛋白质二聚基序的物理化学性质，构象和合作的流动性的调查将是一般的肽/蛋白质折叠和稳定性的兴趣。

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.