The pathways for the generation and isomerization of disulfide bonds during the oxidative folding of proteins destined for secretion are still poorly understood. Reduced RNase and its fully oxidized, but mispaired counterpart, scrambled RNase, remain widely-used model substrates for these studies. However, there is a continuing need for additional convenient substrates that can be used to assess oxidative folding pathways. Here we introduce a bioluminescent assay for oxidative protein folding which utilizes reduced Gaussia princeps luciferase (GLuc). Native GLuc, a 19.9 kD monomeric protein containing 5 disulfides and no free cysteine residues, catalyzes the oxidation of coelenterazine independent of ATP or other cofactors with a burst of intense bioluminescence (at ~470 nm). Procedures have been developed for the quantitative generation of reduced GLuc (rGLuc) that exhibit less than 0.01% of the bioluminescence of the native protein. We show that up to ~80% activity can be recovered in a range of oxidizing environments including glutathione redox buffers, other small molecular oxidants, or nanomolar levels of avian and human Quiescin sulfhydryl oxidase (QSOX1). Scrambled GLuc has also been prepared and examined as a substrate for protein disulfide isomerase (PDI). Finally, we show that the oxidation of rGLuc to generate native-paired GLuc provides a very sensitive method for assessment of the potency of arsenical and heavy metal ion inhibitors of oxidative protein folding.

Gaussia princeps luciferase: A bioluminescent substrate for oxidative protein folding

254th National Meeting (2017)

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