Title
Bifunctional role of leucine 300 of firefly luciferase in structural rigidity
Author
Farzad Yousefi, Farangis Ataei, Mojtaba Mortazavi, Saman Hosseinkhani
Year
2017
Journal
International Journal of Biological Macromolecules
Abstract
Firefly luciferase is susceptible to thermal inactivation, thereby its intracellular half-life decreased. Previous reports indicated that L300R mutation (LRR mutant) in E354R/Arg356double mutant (ERR mutant) from Lampyris turkestanicus luciferase has increased its thermal stability and rigidity through induction of some ionic bonds with Asp 270 and 271. Disruption of the deduced ionic bonds in an ultra-rigid mutant of firefly luciferase did not reverse the flexibility of the protein. In this study, we investigated the effects of this residue to find the truth behind an extraordinary increase in thermal stability and rigidity of luciferase after replacement of leucine 300 by arginine based on previous reports. For this purpose, L300R, L300K and L300E mutations were performed to compare the effects of these mutations on the native firefly luciferase. In spite of increase of intrinsic fluorescence of the mutants a slight increase in thermostability and retention of kinetic properties was observed. Based on our results, we can conclude that L300R mutation in LRR mutant accompanying with alteration in a flexible loop (352–359) increased thermostability and rigidity of luciferase.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Biochemistry