Unfolding of chondroitinase ABC Ι is dependent on thermodynamic driving force by kinetically rate constant-amplitude compensation: A stopped-flow fluorescence study
| Autor: | Bijan Ranjbar, Abolfazl Golestani, S. Akram Shirdel, Khosrow Khalifeh, Khosro Khajeh |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Models Molecular Conformational change Protein Denaturation Protein Conformation Population Kinetics Bioengineering Chondroitin ABC Lyase Applied Microbiology and Biotechnology Biochemistry 03 medical and health sciences Reaction rate constant Phase (matter) Enzyme Stability Thermal stability education education.field_of_study 030102 biochemistry & molecular biology Chemistry Recombinant Proteins Crystallography 030104 developmental biology Amplitude Spectrometry Fluorescence Amino Acid Substitution Biophysics Mutagenesis Site-Directed Unfolded Protein Response Thermodynamics Chemical stability Biotechnology |
| Zdroj: | Enzyme and microbial technology. |
| ISSN: | 1879-0909 |
| Popis: | We had previously investigated the role of a loop on the activity and conformational stability of chondroitinase ABC Ι (cABC Ι) by constructing some representative mutants in which a network interaction around Asp 689 was manipulated. Here we extended our study by measuring the proteolytic resistance, long term and thermal stability as well as unfolding kinetics of these variants. Long term stability data at 4 and 25 °C for 3 weeks indicates that all mutants remain considerably active at 4 °C. Thermoinactivation rates for all variants shows that the wild type (WT) enzyme retained 50% of its activity after 2 min keeping at 40 °C, while L701T, H700N and H700N/L701T as conformationally stabilized variants, have slower inactivation rate. It was also found that compact and thermodynamically stabilized variants are more resistant to tryptolytic digestion. Also, kinetic curves of chemical unfolding of the enzyme variants from stopped-flow fluorescence measurements were best fitted into a three-exponential function with three rate constants and corresponding amplitudes. We found that the energy barrier of the fast unfolding phase is lower in stabilized variants; while the amplitude of this phase to the whole amplitude of the unfolding reaction is lower than that of destabilized variants, indicating more population of stabilized mutants unfold via slower unfolding phase. We concluded that the rate of local conformational change alone is not the same that is expected from global thermodynamic stability; however the corresponding amplitude can compensate the rate constant toward thermodynamic stability. |
| Databáze: | OpenAIRE |
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