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Mutation of conserved residues K329 and R330 on the surface of firefly luciferase: Effect on proteolytic degradation
- Jarchi, Samaneh, Ataei, Farangis, Hosseinkhani, Saman
- International journal of biological macromolecules 2018 v.115 pp. 324-330
- Photinus pyralis, fluorescence, half life, luciferase, mutagenesis, mutants, point mutation, proteolysis, trypsin
- Firefly luciferase is highly susceptible to proteolytic digestion that reduces its half-life and leads to loss in sensitivity. Due to the protease contamination in most in vitro and in vivo environments, it has interest to generate some mutations that may lead to improved susceptibility to digestion. Some important conserved residues (including K206, R213, R218, K329, R330 and R337) on accessible and flexible regions on the surface of Photinus pyralis luciferase have been suggested that susceptible to trypsinolysis. In current study, two mutants (K329I and R330Q) are designed to investigate the impact of these conserved sites on the protease stability and flexibility. This study showed that these mutations did not cause resistance against trypsin digestion. K329I mutant was more susceptible to trypsin, but no difference in the digestion pattern was observed. This point mutation brought about structural flexibility, which revealed by quenching and extrinsic fluorescence. The experimental and theoretical studies demonstrated that R330Q mutagenesis didn't have any noticeable effect on the tryptic sites and flexibility. Moreover, the results of proteolysis experiment showed that the primary sites for trypsin digestion are still exposed after both mutations.