Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, 19716.
Department of Chemistry, University of California at Irvine, Irvine, California, 92697.
Protein Sci. 2018 Aug;27(8):1509-1517. doi: 10.1002/pro.3433. Epub 2018 Jul 18.
Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5-disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.
海肾荧光素酶（GLuc）在没有 ATP 的情况下暴露于腔肠素时会产生强烈的蓝色光爆发。在这里，我们表明，这种含有 5 个二硫键的酶可以作为研究氧化蛋白折叠的简便底物。还原型 GLuc（rGLuc）有 10 个游离半胱氨酸残基，作为荧光素酶完全失活，但在没有蛋白二硫键异构酶（PDI）的外源交换活性的情况下，使用一系列氧化方案可以恢复其 60%以上的生物发光活性，与对照相比。硫氧还蛋白 QSOX1 可以使用 rGLuc 在简单的生物发光平板读取器格式中进行测定。同样，低浓度的 rGLuc 可以被脱氢抗坏血酸、过氧化氢或低得多的四硫代高酸钠浓度氧化。在一系列谷胱甘肽氧化还原缓冲液存在下，rGLuc 的氧化重折叠仅被毫摩尔水平的 PDI 略微加速。这种适度的速率增强可能是由于天然 GLuc 中二硫键连接相对简单所致；反映了每个带有两个二硫键的两个同源结构域，带有一个单个结构域间二硫键。当 GLuc 在变性条件下重新氧化时，产生的乱序蛋白（sGLuc）可用于在没有添加外源硫醇的情况下灵敏地测定还原型 PDI。最后，rGLuc 在体外恢复生物发光活性的一般便利性为评估氧化蛋白折叠抑制剂提供了一种灵敏的方法。
