E50A Mutation Increases the Bioluminescence Activity of picALuc

A miniaturized variant of the artificial luciferase (ALuc), named picALuc, has been generated through the deletion of N- and C-terminal residues in ALuc. Although picALuc is small and active, questions remain regarding its the structural organization and inter-residue interactions in the protein. Here, combining computational analysis and mutational studies, we show that the E50A mutation in picALuc results in an increased bioluminescence activity of the protein. Specifically, we generated a structural model of picALuc using the available structure of the Gaussia luciferase (GLuc) that revealed a 'hole' in the structure due to the deletion of N-terminal alpha-helices. Gaussian-accelerated molecular dynamics (GaMD) simulation revealed a rapid 'compaction' of the picALuc structure during the initial phase of the simulation and a number of residues such as E10, E50, and D94 showed salt bridge interactions. Mutation of the residues E10, E50, and D94 individually to an A revealed increased bioluminescence activity of the E50A mutant, while E10A and D94A mutants showed activities similar to the WT protein in living cells. In vitro assays revealed an increase in the Vmax of the E50A mutant, while Khalf and thermal stability of the mutant remained unchanged. Further, dynamic cross-correlation and principal component analyses of the GaMD simulation trajectories of the WT and the E50A mutant picALuc revealed altered collective dynamics in the protein. Finally, we developed a protein fragment complementation assay using picALuc that allows for the monitoring protein-protein interactions (PPIs) in live cells. We envisage that the brighter picALuc reported here will find broad applicability in developing bioluminescence-based assays.