Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase

Abstract

Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase.

Engineering Small Molecule Switches of Protein Function in Zebrafish Embryos

“…amber-stop codon was placed at site K529 of fLuc (fLuc529TAG-rLuc), where a critical lysine residue orients the substrate and stabilizes the adenylated substrate during catalysis through a hydrogen-bonding interaction with the phosphate oxygen. 31 Caging this lysine blocks enzymatic activity, and small molecule-induced decaging results in full activation of fLuc through the formation of the wild-type active site (Figure 2B). 32 Previous studies of decaging PABK with a panel of phosphines in mammalian cells found that 2-(diphenylphosphinyl)benzamide (2DPBM) showed the fastest reaction rate and no toxicity.…”

“…For the analysis of small molecule control of enzyme function using PABK, we measured the fLuc activity. The amber-stop codon was placed at site K529 of fLuc (fLuc529TAG-rLuc), where a critical lysine residue orients the substrate and stabilizes the adenylated substrate during catalysis through a hydrogen-bonding interaction with the phosphate oxygen . Caging this lysine blocks enzymatic activity, and small molecule-induced decaging results in full activation of fLuc through the formation of the wild-type active site (Figure B) .…”

Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability

“…To study the effect of local conformational constraint on the biochemical properties of luciferase, we investigated the thermal stability of representative luciferases, which has been related to the dynamic behavior of firefly luciferase. 38 As shown in Fig. 6a, incubation of wild-Page 15 of 34 CCS Chemistry type luciferase and luciferase containing 6 at 37 o C in 50 mM Tris-HCl, pH 7.4, for 80 minutes resulted in a steady loss in their ability to produce light, as aliquots were taken as a function of time and then treated with luciferin, ATP and Mg 2+ .…”

Engineering Small Molecule Switches of Protein Function in Zebrafish Embryos

“…The amber stop codon was placed at site K529 of fLuc (fLuc529TAG-rLuc) where a critical lysine residue orients the substrate and stabilizes the adenylated substrate during catalysis through a hydrogen bonding interaction with the phosphate oxygen. 29 Caging this lysine blocks enzymatic activity and small molecule-induced decaging results in full activation of fLuc through formation of the wild-type active site (Figure 2B). 30 Previous studies of decaging PABK with a panel of phosphines in mammalian cells found that 2-(diphenylphosphinyl)benzamide (2DPBM) showed the fastest reaction rate and no toxicity.…”