Abstract

The UnaG protein is a ligand (unconjugated bilirubin) dependent fluorescence protein isolated from Unagi freshwater eel larvae and expressed as fusion in heterologous expression systems. Bilirubin is a tetrapyrrole molecule mainly produced from heme catabolism by the destruction of erythrocytes in the body. Bilirubin can cause kernicterus, a serious condition associated with permanent neurological damage in neonates with the passage of brain tissue. Different methods have been developed for plasma bilirubin analysis and quantification. The use of UnaG fluorescence protein triggered by bilirubin has become a new approach in bilirubin studies. In this study, we aimed to investigate the biophysical characterization of ligand interactions with the proteins obtained as a result of mutations (UnaG^Y99F_Y134W, UnaG^N57E, UnaG^L41F, and UnaG^F17M) on the amino acid sequence of TolAIII-UnaG protein. After the purity levels of the expressed proteins have been analyzed by SDS-PAGE, secondary structures and thermal melting temperatures of the proteins have been examined by circular dichroism spectroscopy. Then determination of excitation and emission points by fluorescence spectroscopy, titration studies have been performed with bilirubin, and dissociation constant was calculated. According to the biophysical characterization studies, UnaG^L41F has the highest affinity and stability among the mutants.

摘要

UnaG 蛋白是一种配体（未结合胆红素）依赖性荧光蛋白，从鳗鱼淡水鳗鱼幼虫中分离出来，并在异源表达系统中融合表达。胆红素是一种四吡咯分子，主要通过体内红细胞的破坏而由血红素分解代谢产生。胆红素可引起核黄疸，这是一种严重的疾病，与脑组织通过时新生儿的永久性神经损伤有关。已经开发了用于血浆胆红素分析和定量的不同方法。利用胆红​​素触发的UnaG荧光蛋白成为胆红素研究的新途径。在这项研究中，我们旨在研究配体与由于突变（UnaG ^Y99F_Y134W，UnaG）获得的蛋白质相互作用的生物物理特征。^N57E、 UnaG ^L41F和 UnaG ^F17M ) 在 TolAIII-UnaG 蛋白的氨基酸序列上。在通过 SDS-PAGE 分析表达蛋白质的纯度水平后，通过圆二色光谱检查蛋白质的二级结构和热熔解温度。然后通过荧光光谱测定激发点和发射点，用胆红素进行滴定研究，并计算解离常数。根据生物物理特性研究，UnaG ^L41F在突变体中具有最高的亲和力和稳定性。