Substitution of His by Asp modify thermal stability of Firefly luciferase

Light emission of luciferase is used in a wide variety of biochemical assays in clinical, industrial and scientific research applications . However, several factors limit further applications of this technology, one of them is the low stability of luciferase enzyme in the room and human physiological temperature. A common strategy to improve thermal stability of proteins is to introduce new salt bridges or increase the number of hydrogen bonds in the solvent exposed flexible region of proteins. By calculating the root mean square fluctuation (RMSF) of backbone atoms at two temperatures 300 and 340 K, thermal sensitive (flexible) regions of luciferase were identified. Histidine461 and histidine489 are located in the flexible regions, therefore we decided to substitute them with a negatively charged residue, aspartate. Substitution of His461 by aspartate in H461D decreased ATP binding affinity, reduced the melting temperature of protein by around 26 °C and shifted its optimum temperature of activity to 10 °C. In line with the common feature of psychrophilic enzymes, the MD data showed that the overall flexibility of H461D was relatively high at low temperature, probably due to a decrease in the number of salt bridges around the mutation site. On the other hand, substitution of His489 by aspartate in 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.

کلیدواژه ها:

Luciferase ، Thermal stability ، Flexibility ، Molecular Dynamics simulation ، Histidine