Replacement of some hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase with arginine increases thermostability of this enzyme. Herein, thermodynamic and kinetic of unfolding reactions of wild type (WT), E354R/356R, E354R/356R-I232R and E354R/356R-Q35R/L182R/I232R variants, has been investigated. Fluorescence and Far-UV circular dichroism measurements using urea as a chemical denaturant indicated that the value of ΔG(H2O) for all variants is greater than that of WT enzyme. Analysis of m-values, as a measure of difference in the solvent accessible surface area between the native and denatured states of protein, revealed that higher stability of mutants is related to their higher degree of compactness in the folded state. Results of unfolding kinetic experiments showed that all variants have three-exponential behavior in which they unfolded with three rate constants and corresponding amplitudes. Increasing the rate constants of fast unfolding phase in mutants relative to WT protein may be attributed to more compactness and more kinetic sensitivity of their folded state to urea. However, more population of WT protein was unfolded from fast unfolding phase. Results of this investigation highlight kinetic stability of luciferase via a slow rate of unfolding.
© 2016 The American Society of Photobiology.