The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Buffers
2.3. Fibril Preparation
2.4. UV Measurements
2.5. Fluorescence Quenching
2.6. Circular Dichroism (CD)
2.7. Differential Scanning Calorimetry (DSC)
3. Results
3.1. HEWL Molecular Structure and Stability Depend on the Buffer Identity
3.2. The Amyloid Fibril Formation
3.3. Electrostatic Screening Facilitates Fibrillization
3.4. Binding Inert Molecules Inhibits HEWL Fibrillization
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
HEWL | Hen egg-white lysozyme |
CD | Circular dichroism |
DSC | Differential scanning calorimetry |
ThT | Thioflavin T |
TRIS | tris(hydroxymethyl)aminomethane |
HEPES | 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid |
ANS | 8-(Phenylamino)-1-naphthalenesulfonic acid |
PEG | Polyethylene glycol |
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pH Value | Net Charge | Positive Amino Acids | Negative Amino Acids |
---|---|---|---|
2.0 | +18.0 | 11 Arg, 6 Lys, 1 His | / |
3.0 | +18.0 | 11 Arg, 6 Lys, 1 His | / |
4.5 | +8.0 | 11 Arg, 6 Lys, 1 His | 7 Asp, 2 Glu, 1 Leu |
7.0 | +7.0 | 11 Arg, 6 Lys | 7 Asp, 2 Glu, 1 Leu |
7.5 | +7.0 | 11 Arg, 6 Lys | 7 Asp, 2 Glu, 1 Leu |
8.0 | +7.0 | 11 Arg, 6 Lys | 7 Asp, 2 Glu, 1 Leu |
9.0 | +7.0 | 11 Arg, 6 Lys | 7 Asp, 2 Glu, 1 Leu |
10.0 | +7.0 | 11 Arg, 6 Lys | 7 Asp, 2 Glu, 1 Leu |
Solution | -Helix | -Antiparallel Sheet | -Parallel Sheet | Turn |
---|---|---|---|---|
0.5 M glycine, pH = 2.0 | 24 | 12 | 3 | 16 |
0.25 M glycine, pH = 2.0 | 29 | 8 | 3 | 11 |
0.5 M glycine, pH = 3.0 | 23 | 15 | 4 | 13 |
0.25 M glycine, pH = 3.0 | 28 | 9 | 2 | 10 |
0.5 M glycine, pH = 9.0 | 28 | 11 | 4 | 15 |
0.25 M glycine, pH = 9.0 | 30 | 10 | 1 | 11 |
0.5 M glycine, pH = 10.0 | 28 | 14 | 2 | 18 |
0.25 M glycine, pH = 10.0 | 31 | 12 | 1 | 13 |
0.5 M TRIS, pH = 7.0 | 24 | 12 | 5 | 14 |
0.25 M TRIS, pH = 7.0 | 28 | 10 | 1 | 11 |
0.5 M TRIS, pH = 7.5 | 25 | 9 | 4 | 16 |
0.25 M TRIS, pH = 7.5 | 29 | 10 | 1 | 13 |
0.5 M TRIS, pH = 8.0 | 25 | 10 | 5 | 15 |
0.25 M TRIS, pH = 8.0 | 29 | 14 | 3 | 14 |
0.5 M TRIS, pH = 9.0 | 31 | 13 | 2 | 17 |
0.25 M TRIS, pH = 9.0 | 30 | 14 | 0 | 14 |
0.25 M cacodylate, pH = 7.0 | 24 | 13 | 5 | 16 |
0.5 M cacodylate, pH = 7.0 | 27 | 15 | 2 | 16 |
0.1 M KCl-HCl, pH = 2.0 | 24 | 11 | 3 | 16 |
0.5 M KCl-HCl, pH = 2.0 | 23 | 12 | 2 | 15 |
0.25 M KCl-HCl, pH = 2.0 | 24 | 15 | 3 | 15 |
0.5 M Phosphate, pH = 2.0 | 30 | 7 | 2 | 15 |
0.5 M Phosphate, pH = 7.0 | 25 | 11 | 4 | 16 |
Solution | -Helix | -Antiparallel Sheet | -Parallel Sheet | Turn |
---|---|---|---|---|
0.5 M glycine (C) | 24 | 12 | 3 | 16 |
0.5 M glycine (S) | 23 | 10 | 5 | 15 |
0.5 M glycine (A) | 2 | 87 | 0 | 11 |
0.25 M glycine (C) | 29 | 8 | 3 | 11 |
0.25 M glycine (S) | 28 | 4 | 6 | 12 |
0.25 M glycine (A) | 33 | 15 | 0 | 12 |
0.1 M KCl-HCl (C) | 24 | 11 | 3 | 16 |
0.1 M KCl-HCl (S) | 22 | 13 | 3 | 15 |
0.1 M KCl-HCl (A) | 28 | 18 | 10 | 11 |
0.25 M KCl-HCl (C) | 24 | 15 | 3 | 15 |
0.25 M KCl-HCl (S) | 21 | 12 | 5 | 16 |
0.25 M KCl-HCl (A) | 0 | 99 | 0 | 2 |
0.5 M KCl-HCl (C) | 23 | 12 | 2 | 15 |
0.5 M KCl-HCl (S) | 19 | 18 | 5 | 14 |
0.5 M KCl-HCl (A) | 0 | 93 | 0 | 7 |
0.5 M phosphate (C) | 30 | 7 | 2 | 15 |
0.5 M phosphate (S) | 28 | 6 | 3 | 16 |
0.5 M phosphate (A) | 30 | 6 | 2 | 15 |
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Brudar, S.; Hribar-Lee, B. The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism. Biomolecules 2019, 9, 65. https://doi.org/10.3390/biom9020065
Brudar S, Hribar-Lee B. The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism. Biomolecules. 2019; 9(2):65. https://doi.org/10.3390/biom9020065
Chicago/Turabian StyleBrudar, Sandi, and Barbara Hribar-Lee. 2019. "The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism" Biomolecules 9, no. 2: 65. https://doi.org/10.3390/biom9020065