Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Reagents
2.2. Preparation of Proteins and Reagents
2.3. Thermal Denaturation Measurements
2.4. Circular Dichroism (CD) Measurements
2.5. Activity Measurements
3. Results
3.1. Thermal Denaturation Study of α-LA
3.2. Analysis of Denaturation Curves of α-LA
3.3. Thermal Denaturation Study of Lysozyme
3.4. Analysis of Denaturation Curves of Lysozyme
3.5. Far- and Near-UV CD Measurements
3.6. Activity Measurements
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
GdmCl, | guanidinium chloride; |
UV, | ultra-violet; |
CD, | circular dichroism; |
Tm, | midpoint of thermal denaturation; |
ΔHm, | enthalpy change at Tm; |
ΔCp, | constant-pressure heat capacity change; |
∆GD°, | Gibbs free energy change at 25 °C; |
Km, | Michaelis constant; |
kcat, | catalytic constant; |
F70, | Ficoll 70; |
D70, | Dextran 70; |
D40, | Dextran 40. |
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[Crowder] (mg mL−1) | Tm (oC) | ∆Hm (kcal mol−1) | ΔCp (kcal mol−1 K−1) | ∆GDo (kcal mol−1) |
---|---|---|---|---|
0 | 42.8 ± 0.2 (42.9 ± 0.2) | 51 ± 3 (51 ± 3) | 1.56 ± 0.09 | 2.07 ± 0.16 (2.08 ± 0.15) |
Ficoll 70 | ||||
50 | 43.4 ± 0.3 | 52 ± 3 | 1.55 ± 0.09 | 2.18 ± 0.21 |
100 | 44.4 ± 0.2 | 52 ± 2 | 1.58 ± 0.06 | 2.22 ± 0.11 |
150 | 45.0 ± 0.3 | 54 ± 2 | 1.57 ± 0.08 | 2.39 ± 0.12 |
200 | 45.4 ± 0.1 | 55 ± 2 | 1.55 ± 0.07 | 2.49 ± 0.11 |
250 | 46.4 ± 0.2 | 57 ± 3 | 1.56 ± 0.07 | 2.67 ± 0.22 |
300 | 47.1 ± 0.3 | 58 ± 4 | 1.57 ± 0.06 | 2.78 ± 0.26 |
350 | 47.4 ± 0.3 (47.5 ± 0.2) | 59 ± 3 (59 ± 2) | 1.57 ± 0.09 | 2.86 ± 0.18 (2.87 ± 0.15) |
Dextran 70 | ||||
50 | 44.1 ± 0.2 | 52 ± 3 | 1.58 ± 0.09 | 2.20 ± 0.19 |
100 | 45.9 ± 0.3 | 53 ± 2 | 1.57 ± 0.07 | 2.37 ± 0.12 |
150 | 47.5 ± 0.2 | 55 ± 2 | 1.58 ± 0.07 | 2.58 ± 0.12 |
200 | 49.1 ± 0.2 | 57 ± 3 | 1.56 ± 0.08 | 2.82 ± 0.20 |
250 | 50.5 ± 0.1 | 59 ± 2 | 1.55 ± 0.06 | 3.04 ± 0.12 |
300 | 51.1 ± 0.2 (51.0 ± 0.2) | 60 ± 2 (60 ± 3) | 1.55 ± 0.09 | 3.15 ± 0.41 (3.15 ± 0.16) |
Dextran 40 | ||||
50 | 44.9 ± 0.3 | 52 ± 2 | 1.57 ± 0.09 | 2.25 ± 0.13 |
100 | 47.8 ± 0.3 | 53 ± 2 | 1.59 ± 0.07 | 2.44 ± 0.12 |
150 | 50.4 ± 0.1 | 55 ± 3 | 1.55 ± 0.08 | 2.73 ± 0.26 |
200 | 52.9 ± 0.2 | 57 ± 4 | 1.58 ± 0.08 | 2.93 ± 0.32 |
250 | 54.0 ± 0.3 | 59 ± 3 | 1.56 ± 0.09 | 3.16 ± 0.29 |
300 | 54.3 ± 0.3 (54.3 ± 0.2) | 64 ± 2 (64 ± 2) | 1.55 ± 0.09 | 3.63 ± 0.16 (3.63 ± 0.16) |
[Dextran 40] (mg mL−1) | Tm (obs.)c (oC) | Tm (corr.)d (oC) | ∆Hm (obs.) c (kcal mol−1) | ∆Hm (corr.) d (kcal mol−1) | ΔGDº (kcal mol−1) |
---|---|---|---|---|---|
pH 7.0 | |||||
0 | 59.9 ± 0.3 (59.8 ± 0.3) | 85.5 ± 0.3 (85.4 ± 0.3) | 93 ± 3 (93 ± 4) | 127 ± 3 (127 ± 4) | 12.80 ± 0.30 (12.75 ± 0.27) |
100 | 60.1 ± 0.3 | 85.7 ± 0.3 | 94 ± 3 | 128 ± 3 | 12.98 ± 0.40 |
150 | 60.4 ± 0.2 | 86.0 ± 0.2 | 95 ± 2 | 129 ± 2 | 13.21 ± 0.26 |
200 | 60.8 ± 0.2 | 86.4 ± 0.2 | 97 ± 3 | 131 ± 3 | 13.57 ± 0.24 |
250 | 61.7 ± 0.2 | 87.3 ± 0.2 | 99 ± 4 | 133 ± 4 | 13.88 ± 0.40 |
300 | 62.0 ± 0.3 (61.9 ± 0.3) | 87.6 ± 0.3 (87.5 ± 0.3) | 100 ± 2 (99 ± 2) | 134 ± 2 (133 ± 2) | 14.06 ± 0.34 (13.89 ± 0.31) |
pH 2.0 | |||||
0 | 57.6 ± 0.2 (57.6 ± 0.2) | - | 84 ± 2 (84 ± 2) | - | 5.61 ± 0.13 (5.61 ± 0.13) |
100 | 58.8 ± 0.2 | - | 86 ± 2 | - | 5.92 ± 0.19 |
150 | 59.5 ± 0.3 | - | 88 ± 2 | - | 6.19 ± 0.23 |
200 | 61.8 ± 0.2 | - | 92 ± 3 | - | 6.78 ± 0.28 |
250 | 62.9 ± 0.3 | - | 94 ± 3 | - | 7.06 ± 0.29 |
300 | 64.1 ± 0.2 (64.0 ± 0.2) | - | 97 ± 2 (96 ± 3) | - | 7.49 ± 0.24 (7.37 ± 0.26) |
[Dextran 40], mg mL−1 | ∆Cp, kcal mol− K−1 | ||||
0 | 1.60 ± 0.09 | ||||
100 | 1.59 ± 0.05 | ||||
150 | 1.58 ± 0.07 | ||||
200 | 1.58 ± 0.06 | ||||
250 300 | 1.59 ± 0.08 1.59 ± 0.07 |
pH | Dextran 40 (300 mg mL-1) | Dextran 70 (300 mg mL-1) | Ficoll 70 (300 mg mL-1) | |||
---|---|---|---|---|---|---|
∆Tm, °C | %∆∆GD° | ∆Tm, °C | %∆∆GD° | ∆Tm, °C | %∆∆GD° | |
7.0 | 11.5 | 75.36 | 8.3 | 52.17 | 4.3 | 34.29 |
6.5 | 9.2 | 36.64 | 7.0 | 31.25 | 3.5 | 20.17 |
6.0 | 6.8 | 18.67 | 5.5 | 17.53 | 2.7 | 13.89 |
5.5 | 4.9 | 15.23 | 4.1 | 14.62 | 2.0 | 12.22 |
pH | Dextran 40 (300 mg mL−1) | Dextran 70 (300 mg mL−1) | Ficoll 70 (300 mg mL−1) | |||
---|---|---|---|---|---|---|
∆Tm, °C | %∆∆GD° | ∆Tm, °C | %∆∆GD° | ∆Tm, °C | %∆∆GD° | |
7.0 | 2.1 | 9.84 | 1.7 | 9.29 | 1.0 | 7.34 |
6.0 | 2.6 | 11.52 | 2.1 | 9.99 | 1.0 | 6.52 |
5.0 | 3.3 | 13.46 | 2.2 | 11.32 | 1.1 | 7.28 |
4.0 | 5.8 | 17.23 | 2.3 | 12.02 | 1.3 | 6.81 |
3.0 | 6.1 | 20.70 | 3.0 | 16.23 | - | - |
2.0 | 6.5 | 33.51 | 4.3 | 25.84 | - | - |
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Shahid, S.; Hasan, I.; Ahmad, F.; Hassan, M.I.; Islam, A. Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder. Biomolecules 2019, 9, 477. https://doi.org/10.3390/biom9090477
Shahid S, Hasan I, Ahmad F, Hassan MI, Islam A. Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder. Biomolecules. 2019; 9(9):477. https://doi.org/10.3390/biom9090477
Chicago/Turabian StyleShahid, Sumra, Ikramul Hasan, Faizan Ahmad, Md. Imtaiyaz Hassan, and Asimul Islam. 2019. "Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder" Biomolecules 9, no. 9: 477. https://doi.org/10.3390/biom9090477
APA StyleShahid, S., Hasan, I., Ahmad, F., Hassan, M. I., & Islam, A. (2019). Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder. Biomolecules, 9(9), 477. https://doi.org/10.3390/biom9090477