Stability and flexibility of Ca2+-regulated phtoproteins: a comparative study

Photoproteins are present in many marine coelenterates and ctenophores allow these organisms to emit light. Photoproteins from ctenophores include mnemiopsin, berovin, bolinopsin and BfosPP while photoproteins such as aequorin, obelin and clytin belong to the coeleterates. Improving the stability of photoproteins is considered as an important purpose for both basic research and analytical applications. In order to reveal the difference in the accessibility and flexibility of these families, apomnemiopsin and apoaequorin expressed in E. coli BL21 (DE3) and then purified. Fluorescence quenching was carried out by acrylamide, KI and CsCl as different quenchers to protein solutions at an excitation wavelength of 280 nm. Quenching data were analyzed in terms of the Stern–Volmer equation. The Stern–Volmer plots indicates that the aromatic residues in aequorin are more exposed to the solvent as compared to the mnemiopsin, which suggests that mnemiopsin is locally more compact. Limited proteolysis was with trypsin for mnemiopsin and aequorin. The SDS-PAGE analysis of the proteolysis mixture during 2 h reaction at 37 ◦C reveals that the digestion was more extensive for aequorin compared to mnemiopsin. Thermodynamic stability of proteins was evaluated in isothermal denaturation experiment using guanidine hydrochloride (GdnHCl) as chemical denaturant. According to thermodynamic data, aequorin has lower stability compared to mnemiopsin. It is noticeable that the value of ∆G (H2O) is related to other thermodynamic parameters including m-value and [GdnHCl]50%. However, lower value of [GdnHCl]50% in aequorin suggests that the interaction of GdnHCl molecules with its residues is more facilitated when compared with mnemiopsin indicating more penetrability of this variant to GdnHCl compared with mnemiopsin. With the aforementioned observations, our research confirms the greater flexibility of aequorin, which suggests that mnemiopsin is more stable than aequorin.