Surface Arginine Saturation Effect on Unfolding Reaction of Firefly Luciferase: A Thermodynamic and Kinetic Perspective

Abstract

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.

Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase

“…Although Fluc is applied to a hygiene monitoring system ( Corbitt et al, 2000 ; Bakke and Suzuki, 2018 ), incubation of the enzyme for 1 h at 37°C resulted in an 83% decrease in signal intensity. To overcome this problem, many researchers have attempted to enhance the stability of Fluc ( White et al, 1996 ; Koksharov and Ugarova, 2011a ; Koksharov and Ugarova, 2011b ; Rasouli et al, 2011 ; Lohrasbi-Nejad et al, 2016 ; Solgi et al, 2016 ; Branchini and Southworth, 2017 ; Jazayeri et al, 2017 ; Rahban et al, 2017 ; Pozzo et al, 2018 ; Branchini et al, 2019 ). In this study, we applied Luciola lateralis (Heike-botaru) Fluc (LlL) with mutations for thermostability and organic solvent tolerance ( Kodama and Nasu, 2011 ) to FlimPIA for improving the stability at physiological reaction condition.…”