Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Preparation of Protein Solutions
2.2.2. Preparation of Working Solution of the Protein
2.2.3. Denaturant Stock Solution Preparations
2.2.4. Preparation of Crowder Stock Solution
2.2.5. Spectral Measurements and Analysis of Isothermal Transition Curves
Absorption Spectroscopy
Far-UV Circular Dichroism Spectroscopy
Analysis of Isothermal Denaturation Curves
2.2.6. Isothermal Titration Calorimetry
2.2.7. Computational Methods
3. Results
3.1. GdmCl- and Urea-Induced Mb Denaturation in the Absence and Presence of Ficoll 70 at Different pH Values
3.1.1. Absorption Measurements
3.1.2. Circular Dichroism Measurements
3.2. GdmCl-Induced and Urea-Induced Denaturation of Mb in the Absence and Presence of Dextran 70 at Different pH Values
3.2.1. Absorption Measurements
3.2.2. Circular Dichroism Measurements
3.3. Interaction Studies
3.3.1. Isothermal Titration Calorimetry Measurements
3.3.2. Molecular Docking Studies
4. Discussions
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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pH | [Ficoll 70] mg/mL | GdmCl-Induced Denaturation | Urea-Induced Denaturation | ||
---|---|---|---|---|---|
ΔGD°(kcal mol−1) | Cm[M] | ΔGD°(kcal mol−1) | Cm[M] | ||
6 | 0 | 10.13 ± 0.11 (10.15 ± 0.12) * | 1.26 ± 0.04 (1.25 ± 0.05) * | 10.25 ± 0.18 (10.23±0.15) * | 3.99 ± 0.05 (4.00 ± 0.04) * |
100 | 9.70 ± 0.18 | 1.15 ± 0.06 | 9.75 ± 0.15 | 3.60 ± 0.03 | |
200 | 9.22 ± 0.12 | 1.00 ± 0.05 | 9.31 ± 0.11 | 3.30 ± 0.04 | |
300 | 8.78 ± 0.13 | 0.95 ± 0.05 | 8.72 ± 0.10 | 3.07 ± 0.06 | |
7 | 0 | 11.10 ± 0.14 (11.13 ± 0.16) * | 1.53 ± 0.03 (1.56 ± 0.02) * | 10.95 ± 0.15 (10.98 ± 0.13) * | 5.10 ± 0.04 (5.09 ± 0.05) * |
100 | 10.34 ± 0.15 | 1.4 ± 0.04 | 10.38 ± 0.16 | 4.63 ± 0.05 | |
200 | 9.87 ± 0.11 | 1.2 ± 0.05 | 9.87 ± 0.14 | 4.15 ± 0.06 | |
300 | 9.08 ± 0.14 | 1.1 ± 0.03 | 9.11 ± 0.13 | 3.58 ± 0.04 | |
8 | 0 | 10.23 ± 0.15 (10.21 ± 0.13) * | 1.82 ± 0.04 (1.81 ± 0.03) * | 10.25 ± 0.11 (10.23 ± 0.14) * | 5.83 ± 0.04 (5.85 ± 0.03) * |
100 | 9.76 ± 0.11 | 1.7 ± 0.03 | 9.71 ± 0.18 | 5.46 ± 0.05 | |
200 | 9.28 ± 0.13 | 1.65 ± 0.04 | 9.28 ± 0.13 | 5.04 ± 0.05 | |
300 | 8.81 ± 0.14 | 1.60 ± 0.05 | 8.81 ± 0.16 | 4.75 ± 0.06 |
pH | [Dextran 70] mg/mL | GdmCl-Induced Denaturation | Urea-Induced Denaturation | ||
---|---|---|---|---|---|
ΔGD°(kcal mol−1) | Cm[M] | ΔGD°(kcal mol−1) | Cm[M] | ||
6 | 0 | 10.13 ± 0.11 (10.15 ± 0.12) * | 1.26 ± 0.04 (1.25 ± 0.05) * | 10.25 ± 0.18 (10.23±0.15) * | 3.99 ± 0.05 (4.00 ± 0.04) * |
100 | 9.85 ± 0.15 (9.85 ± 0.14) * | 1.04 ± 0.02 (1.05 ± 0.03) * | 9.88 ± 0.13 (9.83 ± 0.12) * | 3.60 ± 0.03 (3.65 ± 0.02) * | |
200 | 9.44 ± 0.14 (9.43 ± 0.13) * | 0.98 ± 0.02 (0.98 ± 0.03) * | 9.34 ± 0.14 (9.31 ± 0.12) * | 3.10 ±0.02 (3.13 ± 0.03) * | |
300 | 9.05 ± 0.13 (9.05 ± 0.14) * | 0.85 ± 0.03 (0.85 ± 0.02) * | 9.11 ± 0.13 (9.10 ± 0.14) * | 3.70 ± 0.03 (3.71 ± 0.02) * | |
7 | 0 | 11.10 ± 0.14 (11.13 ± 0.16) * | 1.53 ± 0.03 (1.56 ± 0.02) * | 10.95 ± 0.15 (10.98 ± 0.13) * | 5.10 ± 0.04 (5.09 ± 0.05) * |
100 | 10.55 ± 0.13 (10.57 ± 0.16) * | 1.47 ± 0.02 (1.46 ± 0.03) * | 10.53 ± 0.16 (10.52 ± 0.18) * | 4.71 ± 0.03 (4.70 ± 0.03) * | |
200 | 10.11 ± 0.12 (10.10 ± 0.11) * | 1.35 ± 0.01 (1.34 ± 0.04) * | 10.17 ± 0.14 (10.14 ± 0.13) * | 4.34 ± 0.03 (4.35 ± 0.02) * | |
300 | 9.68 ± 0.13 (9.65 ± 0.12) * | 1.15 ± 0.03 (1.16 ± 0.02) * | 9.72 ± 0.13 (9.69 ± 0.12) * | 4.07 ± 0.02 (4.08 ± 0.03) * | |
8 | 0 | 10.23 ± 0.15 (10.21 ± 0.13) * | 1.82 ± 0.04 (1.81 ± 0.03) * | 10.25 ± 0.11 (10.23 ± 0.14) * | 5.83 ± 0.04 (5.85 ± 0.03) * |
100 | 9.84 ± 0.14 (9.84 ± 0.16) * | 1.58 ± 0.03 (1.59 ± 0.02) * | 9.85 ± 0.13 (9.84 ± 0.15) * | 4.95 ± 0.02 (4.93 ± 0.03) * | |
200 | 9.40 ± 0.11 (9.43 ± 0.13) * | 1.36 ± 0.02 (1.35 ± 0.02) * | 9.42 ± 0.15 (9.45 ± 0.13) * | 4.61 ± 0.02 (4.62 ± 0.03) * | |
300 | 9.03 ± 0.12 (9.01 ± 0.14) * | 1.01 ± 0.03 (1.00 ± 0.02) * | 9.04 ± 0.17 (9.06 ± 0.14) * | 4.46 ± 0.02 (4.44 ± 0.02) * |
Crowder | N | Ka(M−1) | ΔH(cal mol−1) | ΔS(cal K−1 mol−1) | Kd(µM) | ΔG(kcal mol−1) |
---|---|---|---|---|---|---|
Dextran 70 | 0.74 ± 0.04 | 10.6 × 105 (± 0.88 × 105) | −1.95 (± 0.16) | −42.5 (± 0.3) | 9.43 | −6.83 |
Ficoll 70 | [0.71 (± 0.04)] # | [9.42 × 104 (± 1.08 × 104)] # | [-2.93 × 104 (± 0.50 × 104)] # | [−45.4 (± 0.40)] # | [10.62] # | [−15.77] # |
pH | GdmCl-Induced Denaturation | Urea-Induced Denaturation | ||
---|---|---|---|---|
Ficoll 70 | Dextran 70 | Ficoll 70 | Dextran 70 | |
6.0 | 1.37 ± 0.17 | 1.1 ± 0.12 (1.08 ± 0.13) * | 1.58 ± 0.19 | 1.12 ± 0.14 (1.15 ± 0.15) * |
7.0 | 1.43 ± 0.20 | 1.45 ± 0.12 (1.45 ± 0.13) * | 1.26 ± 0.19 | 1.26 ± 0.14 (1.26 ± 0.13) * |
8.0 | 1.6 ± 0.14 | 1.18 ± 0.12 (1.22 ± 0.14) * | 1.62 ± 0.15 | 1.19 ± 0.16 (1.19 ± 0.13) * |
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Nasreen, K.; Parray, Z.A.; Ahamad, S.; Ahmad, F.; Ahmed, A.; Freeh Alamery, S.; Hussain, T.; Hassan, M.I.; Islam, A. Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders. Biomolecules 2020, 10, 490. https://doi.org/10.3390/biom10030490
Nasreen K, Parray ZA, Ahamad S, Ahmad F, Ahmed A, Freeh Alamery S, Hussain T, Hassan MI, Islam A. Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders. Biomolecules. 2020; 10(3):490. https://doi.org/10.3390/biom10030490
Chicago/Turabian StyleNasreen, Khalida, Zahoor Ahmad Parray, Shahzaib Ahamad, Faizan Ahmad, Anwar Ahmed, Salman Freeh Alamery, Tajamul Hussain, Md. Imtaiyaz Hassan, and Asimul Islam. 2020. "Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders" Biomolecules 10, no. 3: 490. https://doi.org/10.3390/biom10030490