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nmrlearner 11-21-2020 08:41 AM

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR.
 
Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR.

http://www.bionmr.com//www.ncbi.nlm....s-npg_logo.gif Related Articles Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR.

Sci Rep. 2020 Nov 18;10(1):20069

Authors: Wu N, Kobayashi N, Tsuda K, Unzai S, Saotome T, Kuroda Y, Yamazaki T

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 C?-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.


PMID: 33208800 [PubMed - in process]



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