Abstract
Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36–81, 96–145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.
Highlights
Gaussia luciferase (GLuc) contains 10 cysteines, and previous studies demonstrated that the natively folded GLuc contains five disulfide bonds
We showed that by attaching a Solubility Enhancement Petide (SEP) tag containing nine aspartic acids to GLuc’s C-terminus, we could increase the solubility of GLuc, resulting in a spontaneous refolding and the formation of native disulfide bonds
The final yield of GLuc after tags cleavage and two times High-Performance Liquid Chromatography (HPLC) purification (Supplementary Fig. 1) was 1.5 mg per liter of M9 minimal medium culture, which was sufficient for NMR analysis
Summary
GLuc contains 10 cysteines, and previous studies demonstrated that the natively folded GLuc contains five disulfide bonds. The presence of 5 disulfide bonds increases the risks of misfolding when GLuc is bacterially produced, resulting in a low yield[13]. 2D 1H–15N HSQC spectrum of GLuc. The peak assignments are shown using the one-letter code followed by the residue number. Resonance assignments are numbered starting at the first residue (lysine) behind the secretion tag, which was removed without affecting the bioluminescence activity. We showed that by attaching a SEP tag containing nine aspartic acids to GLuc’s C-terminus, we could increase the solubility of GLuc, resulting in a spontaneous refolding and the formation of native disulfide bonds. We used the SEP-tag fused GLuc construct to produce a sufficient amount of 15N and 13C uniformly labeled GLuc for NMR studies. The three-dimensional structure calculated by using CYANA (ver 3.9823) were determined with a backbone ( Cα) RMSD of 1.39Å ± 0.39Å (excluding residues in the flexible regions)
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