Substitution of His by lysine and aspartate in the flexible fragment of Luciferase

Surface salt bridges and enhanced number of hydrogen bonds are believed to increase protein thermal stability. Thermal stability is a property of a protein which measures its ability to preserve its structure at elevated temperatures. Light emission of firefly luciferase is used in a wide variety of biochemical assays in industrial and clinical applications. Enhancement of thermal stability of luciferase is required for bioluminescence applications. In the present study, molecular dynamics (MD) simulation was carried out to assess the molecular flexibility of firefly luciferase. Afterward, thermal stability of luciferase was quantified using enzymatic, spectroscopic and differential scanning calorimetry (DSC) techniques. RMSFs data at two temperatures of 300 and 340 K showed that His489 located in the flexible region. Therefore, we designed two protein mutants H489K and H489D to investigate alteration of salt bridge and hydrogen bond propensities. Substitution of His489 by aspartate (H489D) introduced a new salt bridge between the C-terminal and N-terminal domains and increased protein rigidity but only slightly improved its thermal stability. Histidine substitution by lysine (H489K) change the structure of luciferase around the mutation site. The optimum temperature of enzyme activity of H489K is improved to 30 ºC. Intrinsic and ANS fluorescence studies also demonstrated that little structural changes for these two mutate proteins were occurred. DSC data showed that melting temperature (Tm) of H489D is slightly more than H489K and native form of firefly luciferase. However, our results represent that increasing the number of salt bridges and hydrogen bonds can improve thermal stability of H489D and H489K slightly.