Volume 9, Issue 3 (2018)                   JMBS 2018, 9(3): 417-425 | Back to browse issues page

XML Persian Abstract Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Solgi Z, Khalifeh K, Hosseinkhani S, Ranjbar B. Comparison of Thermodynamic Stability and Kinetic Refolding of Lampyris turkestanicus and Some of Its Mutants. JMBS 2018; 9 (3) :417-425
URL: http://biot.modares.ac.ir/article-22-24434-en.html
1- Biophysics Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran
2- Biology Department, Sciences Faculty, University of Zanjan, Zanjan, Iran
3- Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran
4- Biophysics Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran, Tarbiat Modares University, Nasr Bridge, Jalal-Al-Ahmad Highway, Tehran, Iran , ranjbarb@modares.ac.ir
Abstract:   (4433 Views)
Aims: The probability of establishing electrostatic interactions due to the abundance of charged hydrophilic residues and especially arginine is considered the most important thermal stabilizing factor of thermophilic enzymes. The current study was conducted with the aim of comparing thermodynamic stability and kinetic refolding of Lampyris turkestanicus and some of its mutants.
Materials and Methods: In the present experimental thermal stability and the way of refolding Lampyris turkestanicus and 3 mutations, including ERR, ERR/I232R, ERR/Q35R/I182R/I232R were investigated by various spectroscopic techniques. In order to high expression of proteins, a single clone of each sample was selected and inoculated into 10ml of LB culture medium, containing Kanamycin at a concentration of 50μg/mg and incubated at 37°C with an ideal aeration for 12-15 hours. The culture medium was centrifuged for 5 minutes at 5000g at 4°C to provide the cellular contents of the bacteria. The results were obtained through spectroscopic methods of remote and near circular dichroism, intrinsic fluorescence, differential scanning calorimetry, and kinetics experiments, using fluorescence-stopped flow technique.
Findings: Along with the increase in the number of arginine residues at the protein level, the stability and structural compression of the mutated enzymes in comparison with the wild enzyme were increased and the thermograms obtained from differential scanning calorimetry showed a slight increase in Tm and calorimetric enthalpy of mutated proteins in comparison with wild protein.
Conclusion: The rate constant of refolding mutated enzymes has increased compared with the wild type. The improvement of thermodynamic and kinetic parameters results from the improvement of electrostatic interactions, which results in a higher degree of compression and structural density.
Full-Text [PDF 903 kb]   (3016 Downloads)    
Subject: Agricultural Biotechnology
Received: 2016/10/23 | Accepted: 2017/09/27 | Published: 2018/09/22

1. Xiao L, Honig B. Electrostatic contributions to the stability of hyperthermophilic proteins. J Mol Biol. 1999;289(5):1435-44. [Link] [DOI:10.1006/jmbi.1999.2810]
2. Kumar S, Tsai CJ, Nussinov R. Factors enhancing protein thermostability. Protein Eng. 2000;13(3):179-91. [Link] [DOI:10.1093/protein/13.3.179]
3. Kawashima T, Amano N, Koike H, Makino S, Higuchi S, Kawashima-Ohya Y, et al. Archaeal adaptation to higher temperatures revealed by genomic sequence of Thermoplasma volcanium. Proc Natl Acad Sci U S A. 2000;97(26):14257-62. [Link] [DOI:10.1073/pnas.97.26.14257]
4. Kumar S, Nussinov R. Ion pairs and their stabilities fluctuate in NMR the Bacillus caldolyticus cold shock protein. J Mol Biol. 2001;297:975-88. [Link]
5. Fersht AR. Protein folding and stability: The pathway of folding of barnase. FEBS Lett. 1993;325(1-2):5-16. [Link] [DOI:10.1016/0014-5793(93)81405-O]
6. Fersht AR. Characterizing transition states in protein folding: An essential step in the puzzle. Curr Opin Struct Biol. 1995;5(1):79-84. [Link] [DOI:10.1016/0959-440X(95)80012-P]
7. López-Hernéndez E, Serrano L. Structure of the transition state for folding of the 129 aa protein CheY resembles that of a smaller protein, CI-2. Fold Des. 1996;1(1):43-55. [Link] [DOI:10.1016/S1359-0278(96)00011-9]
8. Mortezavi M, Hosseinkhani S. Design of thermostable luciferases through arginine saturation in solvent-exposed loops. Protein Eng Des Sel. 2011;24(12):893-903. [Link] [DOI:10.1093/protein/gzr051]
9. Mortazavi M, Hosseinkhani S, Khajeh K, Ranjbar B, Emamzadeh AR. Spectroscopic and functional characterization of Lampyris turkestanicus luciferase: A comparative study. Acta Biochim Biophys Sin (Shanghai). 2008;40(5):365-74. [Link] [DOI:10.1111/j.1745-7270.2008.00411.x]
10. Ganjalikhany MR, Ranjbar B, Hosseinkhani S, Khalifeh K, Hassani L. Roles of trehalose and magnesium sulfate on structural and functional stability of firefly luciferase. J Mol Catal B Enzym. 2010;62(2):127-32. [Link] [DOI:10.1016/j.molcatb.2009.09.015]
11. Hosseinkhani S. Molecular enigma of multicolor bioluminescence of firefly luciferas. Cell Mol Life Sci. 2011;68(7):1167-82. [Link] [DOI:10.1007/s00018-010-0607-0]
12. Tisi LC, White PJ, Squirrell DJ, Murphy MJ, Lowe CR, Murray JAH. Development of a thermostable firefly luciferase. Anal Chim Acta. 2002;457(1):115-23. [Link] [DOI:10.1016/S0003-2670(01)01496-9]
13. Hirokowa K, Kajiyama N, Murakami S. Improved practical usefulness of firefly luciferase by gene chimerization and random mutagenesis. Biochim Biophys Acta. 2002;1597(2):271-9. [Link] [DOI:10.1016/S0167-4838(02)00302-3]
14. Law GH, Gandelman OA, Tisi LC, Lowe CR, Murray JA. Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance. Biochem J. 2006;397(2):305-12. [Link] [DOI:10.1042/BJ20051847]
15. Khalifeh K, Ranjbar B, Alipour BS, Hosseinkhani S. The effect of surface charge balance on thermodynamic stability and kinetics of refolding of firefly luciferase. BMB Rep. 2011;44(2):102-6. [Link] [DOI:10.5483/BMBRep.2011.44.2.102]
16. Mehrabi M, Hosseinkhani S, Ghobadi S. Stabilization of firefly luciferase against thermal stress by osmolytes. Int J Biol Macromol. 2008;43(2):187-91. [Link] [DOI:10.1016/j.ijbiomac.2008.05.001]
17. Arakawa T, Timasheff SN. The stabilization of proteins by osmolytes. Biophys J. 1985;47(3):411-4. [Link] [DOI:10.1016/S0006-3495(85)83932-1]
18. White PJ, Squirrell DJ, Arnaud P, Lowe CR, Murray JA. Improved thermostability of the North American firefly luciferase: Saturation mutagenesis at position 354. Biochem J. 1996;319(Pt 2):343-50. [Link] [DOI:10.1042/bj3190343]
19. Said Alipour B, Hosseinkhani S, Ardestani SK, Moradi A. The effective role of positive charge saturation in bioluminescence color and thermostability of firefly luciferase. Photochem Photobiol Sci. 2009;8(6):847-55. [Link] [DOI:10.1039/b901938c]
20. Maghami P, Ranjbar B, Hosseinkhani S, Ghasemi A, Moradi A, Gill P. Relationship between stability and bioluminescence color of firefly luciferase. Photochem Photobiol Sci. 2010;9(3):376-83. [Link] [DOI:10.1039/b9pp00161a]
21. Noble JE. Quantification of protein concentration using UV absorbance and Coomassie dyes. Methods Enzymol. 2014;536:17-26. [Link] [DOI:10.1016/B978-0-12-420070-8.00002-7]
22. Ranjbar B, Gill P. Circular dichroism techniques: Biomolecular and nanostructural analyses - a review. Chem Biol Drug Des. 2009;74(2):101-20. [Link] [DOI:10.1111/j.1747-0285.2009.00847.x]
23. Rezaei Tavirani M, Moghaddamnia SH, Ranjbar B, Amani M, Marashi SA. Conformational study of human serum albumin in pre-denaturation temperatures by differential scanning calorimetry, circular dichroism and UV spectroscopy. J Biochem Mol Biol. 2006;39(5):530-6. [Link] [DOI:10.5483/BMBRep.2006.39.5.530]
24. Khalifeh K, Ranjbar B, Khajeh K, Naderi Manesh H, Sadeghi M, Gharavi S. A stopped-flow fluorescence study of the native and modified lysozyme. Biologia. 2007;62(3):258-64. [Link] [DOI:10.2478/s11756-007-0045-0]
25. Gill P, Tohidi Moghadam T, Ranjbar B. Differential scanning calorimetry techniques: Applications in biology and nanoscience. J Biomol Tech. 2010;21(4):167-93. [Link]
26. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling. Bioinformatics. 2006;22(2):195-201. [Link] [DOI:10.1093/bioinformatics/bti770]
27. DeLuca M, McElroy WD. Kinetics of the firefly luciferase catalyzed reactions. Biochemistry. 1974;13(5):921-5. [Link] [DOI:10.1021/bi00702a015]
28. Zhou XX, Wang YB, Pan YJ, Li WF. Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins. Amino Acids. 2008;34(1):25-33. [Link] [DOI:10.1007/s00726-007-0589-x]
29. Chakravarty S, Varadarajan R. Elucidation of determinants of protein stability through genome sequence analysis. FEBS Lett. 2000;470(1):65-9. [Link] [DOI:10.1016/S0014-5793(00)01267-9]
30. Pace CN, Alston RW, Shaw KL. Charge-charge interactions influence the denatured state ensemble and contribute to protein stability. Protein Sci. 2000;9(7):1395-8. [Link] [DOI:10.1110/ps.9.7.1395]
31. Protasevich I, Ranjbar B, Lobachov V, Makarov A, Gilli R, Briand C, et al. Conformation and thermal denaturation of apocalmodulin: Role of electrostatic mutations. Biochemistry. 1997;36(8):2017-24. [Link] [DOI:10.1021/bi962538g]

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.