Longer characteristic wavelength in a novel engineered photoprotein Mnemiopsin 2.

We designed two mutants of photoprotein Mnemiopsin 2 (Mn2) including M52I and V144I, where the mutations were applied in the EF-hand loops I and III. Far-UV CD measurements demonstrated that the stability of the helices in the wild-type (WT) protein is greater compared with the mutants. Heat-induced denaturation experiments in the apo-form of photoproteins showed that WT Mn2 has higher value of the enthalpy change for the unfolding process, indicating that it has more stabilizing interaction compared with mutants. According to the activity measurement data, both mutants, particularly V144I have lower initial intensity as well as slower decay rate as compared with the WT photoprotein. Importantly, it was found that V144I variant shows 25 nm of red shift in the characteristic wavelengths as compared with the WT photoprotein. This finding can be considered as an advantage for in vivo application of photoprotein for imaging purposes. It concluded that this position on loop III of Mn2 is a hotspot point for characteristic wavelength determination. However, further research on this mutant is needed for making stable variants of Mn2 with novel optical features.