The structural origin and biological function of pH-sensitivity in firefly luciferases

Area of Science:

• Biochemistry
• Molecular Biology
• Bioluminescence Research

Background:

• Firefly luciferases are pH-sensitive, showing spectral shifts at acidic pH, high temperatures, and with heavy metals.
• Other beetle luciferases (click beetles, railroadworms) are pH-insensitive, lacking these spectral shifts.
• The molecular basis for firefly luciferase pH-sensitivity remains largely unexplained despite extensive research.

Purpose of the Study:

• To revise and understand the origin of pH-sensitivity in firefly luciferases.
• To identify specific amino acid residues and structural elements responsible for pH-sensitivity.
• To explore the potential biological importance and applications of pH-sensitive luciferases.

Main Methods:

• Comparative sequence analysis of pH-sensitive and pH-insensitive luciferases.
• Site-directed mutagenesis to alter specific amino acid residues.
• Molecular modeling studies to visualize structural impacts.
• Analysis of bioluminescence spectra under varying conditions.

Main Results:

• Identified specific amino acid substitutions influencing pH-sensitivity and bioluminescence color.
• Localized critical residue changes to a loop between residues 223-235 (Photinus pyralis).
• Discovered a network of hydrogen bonds and salt bridges (N229-S284-E311-R337) crucial for color modulation.
• Proposed that these elements affect the luciferin-binding site, specifically the benzothiazolyl moiety.

Conclusions:

• Structural elements, including a specific amino acid network, are key to firefly luciferase pH-sensitivity and color variation.
• Residual red light emission in pH-sensitive luciferases might be a biologically relevant vestige.
• pH-sensitivity in firefly luciferases holds potential for intracellular biosensing applications.