Photoisomerization of the trans and cis isomers of azobenzene derivatives has been used to control the function of biomolecules in a reversible and nondestructive manner. In this study, affibody molecules, representing a class of small, helical proteins that can be engineered for binding to a wide range of target proteins, have been investigated by the incorporation of a photoswitchable azobenzene derivative in the molecule. Three different Z domain variants were produced by solid phase peptide synthesis and conjugated by thiol-directed chemistry to an azobenzene-based photoswitch. The proteins were screened for binding to and light elution from an IgG-sepharose affinity column. One of the tested Z variants, ZC3, showed efficient binding to the column and could be eluted by irradiation with light at 400 nm. In a reverse affinity chromatography assay, where the ZC3 variant was coupled to sepharose, human IgG1 could be captured to the column and partially eluted by light. Further studies of the azobenzene-conjugated ZC3 domain by surface plasmon resonance (SPR) confirmed the high affinity binding to IgG, and circular dichroism (CD) spectroscopy showed that the protein has a high α-helical secondary structure content.

中文翻译：

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蛋白A衍生的Z结构域与免疫球蛋白G之间的光控可逆结合。

偶氮苯衍生物的反式和顺式异构体的光异构化已被用于以可逆和非破坏性的方式控制生物分子的功能。在这项研究中，通过在分子中掺入可光转换的偶氮苯衍生物，研究了代表一类小的螺旋蛋白的亲和分子，这些蛋白可以被工程化以与多种靶蛋白结合。通过固相肽合成产生了三个不同的Z结构域变体，并通过巯基定向化学将其偶联到基于偶氮苯的光开关上。筛选蛋白质与IgG-琼脂糖亲和柱的结合并从其上轻洗脱。测试的Z变体之一ZC3显示出与色谱柱的有效结合，可以用400 nm的光照射洗脱。在ZC3变异体与琼脂糖偶联的反向亲和色谱分析中，人IgG1可以被捕获到色谱柱上，并被光部分洗脱。通过表面等离振子共振（SPR）对偶氮苯共轭的ZC3结构域的进一步研究证实了与IgG的高亲和力结合，并且圆二色性（CD）光谱表明该蛋白具有较高的α-螺旋二级结构含量。