Surface charge modification increases firefly luciferase rigidity without alteration in bioluminescence spectra

Area of Science:

• Biochemistry
• Protein Engineering
• Enzymology

Background:

• Protein engineering offers strategies for enhancing protein thermostability.
• Surface-exposed loops are key targets for modifying protein stability.
• Lampyris turkestanicus luciferase is a bioluminescent enzyme with potential for engineering.

Purpose of the Study:

• To engineer thermostable Lampyris turkestanicus luciferase variants.
• To investigate the effect of arginine substitutions on luciferase thermal stability.
• To explore the impact of these mutations on enzyme activity and spectral properties.

Main Methods:

• Site-directed mutagenesis was used to introduce arginine substitutions in surface-exposed loop residues.
• Enzyme activity assays were performed at various temperatures to determine thermostability and optimal temperature.
• Homology modeling and molecular docking were employed to analyze the structural effects of mutations.

Main Results:

• Mutant luciferases with arginine substitutions (-I232R, double, and triple mutants) showed significantly increased relative activity at 40°C.
• The optimal temperature for these mutants increased by up to 15°C compared to the wild-type.
• Arginine substitutions did not alter bioluminescence emission spectra or significantly affect specific enzyme activities.

Conclusions:

• Arginine substitution in specific surface-exposed loop residues is an effective strategy for improving luciferase thermostability.
• The engineered mutants exhibit enhanced thermal properties without compromising essential enzymatic functions.
• This study provides valuable insights for designing thermostable enzymes for diverse biological applications.