title: Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase.
abstract: Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D(474) and K(445) accompanying with some H-bond deletion may be involved in destabilization of D474K mutant.
authors.affiliation: Department of Biochemistry, Tarbiat Modares University, Tehran, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Amini-Bayat
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
authors.forename: Rahim
authors.initials: R
authors.lastname: Jafari
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology*; D006860:Hydrogen Bonding; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013057:Spectrum Analysis
chemical_list: D011994:Recombinant Proteins; C485197:luciferase, Photinus; D000249:Adenosine Monophosphate; C044533:3'-(1-butylphosphoryl)adenosine; D049409:Luciferases, Firefly