Engineering aequorin to improve thermostability through rigidifying flexible sites

Abstract

Abstract The photoprotein aequorin has been widely used as a bioluminescent label in various biological and analytical techniques. Here, rigidifying flexible sites as a protein engineering approach was used to design the aequorin variants with high thermostability. Based on two simple approaches, B-factor analysis and replacement of glycine residues in α-helix, three mutations were created in the Gly14 and Glu156 positions. The studies showed that the bioluminescence activity of the G14A mutant was slightly increased, however, it was decreased for the E156N mutant. Also, the G14N mutant showed no significant changes compared to the wild type. Structural studies indicated that E156N mutation became much more flexible than the wild type, although G14A and G14N didn t significantly differ. Irreversible thermoinactivation experiments were done upon incubation of aequorin variants at representative temperatures ranging from 333 to 348 K. The inactivation rate constants (kinact) at 333 K were determined to be 0.009 min−1, 0.01 min−1, and 0.013 min−1 for wild type, G14N, and E156N variants respectively. In contrast, a value of 0.004 min−1 was observed for the G14A mutant. Detailed analysis of the inactivation process showed that the greater thermostability of the G14A mutant is due to the higher activation energy (Ea) and subsequently more values of ∆H‡ and ∆G‡ at a given temperature. However, E156N mutation decreased the thermostability of apoaequorin, and the G14N mutant showed no significant changes. According to molecular dynamics simulation, it seems that the higher thermostability of G14A mutant is due to a local increase in the number of van der Waals interactions, when compared the wild type. Moreover, E156N mutation decreased the thermal stability by increasing flexibility in the C terminal region and losing some contacts of this fragment with other parts of the protein.