METHODS AND APPLICATIONS Gaussia princeps luciferase: A bioluminescent substrate for oxidative protein folding Tiantian Yu, 1 Joanna R. Laird, 2 Jennifer A. Prescher, 2 and Colin Thorpe 1 * 1 Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 2 Department of Chemistry, University of California at Irvine, Irvine, California 92697 Received 20 March 2018; Accepted 23 April 2018 DOI: 10.1002/pro.3433 Published online 25 April 2018 proteinscience.org Abstract: 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% biolu- minescence 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 sulfhy- dryl 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 disul- fide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing condi- tions 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. Keywords: bioluminescence assay; disulfide; Gaussia luciferase; oxidative protein folding; protein disulfide isomerase; quiescin sulfhydryl oxidase; redox buffer; thiol-disulfide exchange; thiol oxidation Additional Supporting Information may be found in the online version of this article. Broader statement: Here we study protein folding coupled to the formation of disulfide crosslinks using a luciferase from the marine shrimp Gaussia princeps. Scission of its five disulfide bonds by chemical reduction abolishes its ability to generate light and permits the conditions that facilitate oxidative protein folding to be studied by following the regain of bioluminescence activity. Gaus- sia luciferase represents a convenient and sensitive new tool to assist in the identification of inhibitors of oxidative protein folding. Grant sponsors: NIH; Grant numbers: 5P30 GM110758, GM26643, P20 GM104316NSF; CBET-1351302 and DGE-1144901. *Correspondence to : [Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716]. E-mail: cthorpe@ udel.edu Published by Wiley V C 2018 The Protein Society PROTEIN SCIENCE 2018 VOL 27:1509—1517 1509