E50A Mutation Increases the Bioluminescence Activity of picALuc

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Artificial luciferase (ALuc) variants are crucial tools in biological research.
• A miniaturized variant, picALuc, was created but its structural dynamics and interactions needed further investigation.
• Understanding picALuc's structure-function relationship is key to optimizing its use in assays.

Purpose of the Study:

• To elucidate the structural organization and inter-residue interactions of picALuc.
• To investigate the impact of specific mutations on picALuc's bioluminescence activity and dynamics.
• To develop an improved picALuc for protein-protein interaction studies.

Main Methods:

• Computational modeling and Gaussian-accelerated molecular dynamics (GaMD) simulations.
• Site-directed mutagenesis (E10A, E50A, D94A) and bioluminescence activity assays (in vitro and in living cells).
• Dynamic cross-correlation and principal component analyses, protein fragment complementation assays.

Main Results:

• A structural model of picALuc revealed a 'hole' due to N-terminal deletions.
• GaMD simulations showed structural compaction and identified key salt bridge interactions (E10, E50, D94).
• The E50A mutation significantly increased bioluminescence activity (higher Vmax) without affecting Khalf or thermal stability, and altered protein dynamics.

Conclusions:

• The E50A mutation enhances picALuc's bioluminescence, making it a brighter reporter.
• Computational and mutational analyses revealed key residues and dynamics influencing picALuc activity.
• A novel protein fragment complementation assay using enhanced picALuc enables live-cell protein-protein interaction monitoring.