Volume 9, Issue 3 (2018)
JMBS 2018, 9(3): 417-425 |
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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
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
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 ,
Abstract: (6628 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.
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.
Keywords: Luciferase, Differential Scanning Calorimetry, Circular Dichroism, Stopped-flow Fluorescence, Thermodynamics, Kinetics
Subject:
Agricultural Biotechnology
Received: 2016/10/23 | Accepted: 2017/09/27 | Published: 2018/09/22
Received: 2016/10/23 | Accepted: 2017/09/27 | Published: 2018/09/22
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