title: The role of proline substitutions within flexible regions on thermostability of luciferase.
abstract: Improving the stability of firefly luciferase has been a critical issue for its wider industrial applications. Studies about hyperthermophile proteins show that flexibility could be an effective indicator to find out weak spots to engineering thermostability of proteins. However, the relationship among flexibility, activity and stability in most of proteins is unclear. Proline is the most rigid residue and can be introduced to rigidify flexible regions to enhance thermostability of proteins. We firstly apply three different methods, molecular dynamics (MD) simulation, B-FITTER and framework rigidity optimized dynamics algorithm (FRODA) to determine the flexible regions of Photinus pyralis luciferase: Fragment 197-207; Fragment 471-481 and Fragment 487-495. Then, introduction of proline is used to rigidify these flexible regions. Two mutants D476P and H489P within most flexible regions are finally designed. In the results, H489P mutant shows improved thermostability while maintaining its catalytic efficiency compared to that of wild type luciferase. Flexibility analysis confirms that the overall rigidity and local rigidity of H489P mutant are greatly strengthened. D476P mutant shows decreased thermosatbility and the reason for this is elucidated at the molecular level. S307P mutation is randomly chosen outside the flexible regions as a control. Thermostability analysis shows that S307P mutation has decreased kinetic stability and enhanced thermodynamic stability.
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, PR China; Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, PR China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin PR China.
authors.forename: Haoran
authors.initials: H
authors.lastname: Yu
authors.affiliation: National Institutes for Food and Drug Control (NIFDC), Beijing 100050, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, PR China; Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, PR China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin PR China.
authors.forename: Chao
authors.initials: C
authors.lastname: Guo
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, PR China; Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, PR China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin PR China.
authors.forename: Yiru
authors.initials: Y
authors.lastname: Gan
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, PR China; Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, PR China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin PR China. Electronic address: huang@tju.edu.cn.
authors.forename: He
authors.initials: H
authors.lastname: Huang
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013696:Temperature; D013997:Time Factors
chemical_list: C485197:luciferase, Photinus; D011392:Proline; D049409:Luciferases, Firefly
keywords: Flexibility; Luciferase; Proline; Rational design; Thermostability