An investigation on the effect of circular permutation on the structure and stability of Renilla luciferase

Protein engineering is an approach to design the new proteins with new structure and functions. One strategy in this field is circular permutation. In this method the C terminal and N terminal of a protein are replaced with each other, so the protein will be inactive. Following a proteolytic cleavage, the structure will back to the first state and protein will be active. Recently, we have designed a circularly permuted form of Renilla luciferase-8 (RLuc8), named CP-RLuc8 as a biosensor for detecting the caspase-9 activity. In this study, we investigated the effect of circular permutation on the structure and stability of Renilla luciferase using fluorescent spectroscopy and circular dichroism (CD). The intrinsic and extrinsic fluorescent studies showed not only the reduced emission intensities for CP-RLuc8 but also a blue-shift was seen in the CP-RLuc8 spectrum in comparison with RLuc8. The result of long time stability indicated that intensity of fluorescence spectroscopy starts to decrease after two days incubation at room temperature and considerable change occurs in the intensity after eight days. Fluorescence intensity of CP-RLuc8 changes after eight days incubation in the room temperature. Thermal stability study for both of proteins, revealed decreased fluorescence intensity with increasing temperature. Notably, the decreasing trend was more about RLuc8 whereas the intensity for CP-RLuc8 was overall lower than its native form. Denaturation study with GdnHCl implied that chemical stability of RLuc8 is different from CP-RLuc8. Acrylamide quenching experiments showed that Stern-Volmer constant for RLuc8 is more than that for CP-RLuc8. Circular dichroism also emphasizes on structural changes in the secondary structure of Renilla luciferase-8 due to replacing the C terminal and N terminal of the protein. Consequently, circular permutation of Renilla luciferase influences its structure and stability remarkably.