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Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the
- Source :
- Biochemistry
- Publication Year :
- 2019
-
Abstract
- The dazzling yellow-green light emission of the common North American firefly Photinus pyralis and other bioluminescent organisms has provided a wide variety of prominent research applications like reporter gene assays and in vivo imaging methods. While the P. pyralis enzyme has been extensively studied, only recently has a second Photinus luciferase been cloned from the species scintillans. Even though the enzymes share very high sequence identity (89.8%), the color of the light they emit, their specific activity, and their stability to heat, pH, and chemical denaturation are quite different with the scintillans luciferase being generally more resistant. Through the construction and evaluation of the properties of chimeric domain swapped, single point, and various combined variants, we have determined that only 6 amino acid changes are necessary to confer all of the properties of the scintillans enzyme to wild-type P. pyralis luciferase. Altered stability properties were attributed to four of the amino acid changes (T214N/S276T/H332N/E354N) and single mutations each predominantly changed emission color (Y255F) and specific activity (A222C). Results of a crystallographic study of the P. pyralis enzyme containing the 6 changes (Pps6) provide some insight into the structural basis for some of the documented property differences.
- Subjects :
- Hot Temperature
Protein Conformation
Genetic Vectors
Fireflies
Spectrometry, X-Ray Emission
Hydrogen-Ion Concentration
Crystallography, X-Ray
Ligands
Article
Luciferases, Firefly
Mutagenesis
Catalytic Domain
Enzyme Stability
Mutation
Escherichia coli
Animals
Mutant Proteins
Amino Acids
Crystallization
Guanidine
Subjects
Details
- ISSN :
- 15204995
- Volume :
- 58
- Issue :
- 42
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.pmid..........0bad9b356ee5861c4258940107524863