| Popis: |
NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. |
