Enantiomeric Effect of d-Amino Acid Substitution on the Mechanism of Action of α-Helical Membrane-Active Peptides
Abstract
:1. Introduction
2. Results
2.1. Peptide Design
2.2. Peptide Secondary Structure
2.3. Peptide Hydrophobicity
2.4. Biological Activity
2.5. Cell Membrane Permeabilization
2.6. Interaction of Peptides with Liposome Model Membranes
2.7. AFM Imaging
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Peptide Synthesis and Purification
4.3. Circular Dichroism (CD) Analysis
4.4. Antimicrobial Activity Assay
4.5. Cell Culture and Analysis
4.6. Hemolytic Activity Assay
4.7. Outer Membrane Permeability Assay
4.8. Inner Membrane Permeability Assay
4.9. Preparation of Large Unilamellar Vesicles (LUVs)
4.10. Tryptophan Fluorescence and Quenching Assay
4.11. AFM Imaging
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
MAPs | cationic membrane-active peptides |
AFM | atomic force microscopy |
KP | potassium phosphate |
TFE | trifluoroethanol |
RP-HPLC | reversed-phase HPLC |
MIC | minimal inhibitory concentration |
MHC | minimal hemolytic concentration |
Chol | cholesterol |
PC | chicken egg phosphatidylcholine |
PS | porcine brain phosphatidylserine |
PE | linear dichroism |
PG | E. coli phosphatidylglycerol |
SM | porcine brain sphingomyelin |
TFA | trifluoroacetic acid |
CD | Circular Dichroism |
MH | Mueller–Hinton |
ONPG | o-nitrophenyl-β-d-galactoside |
NPN | 1-N-phenylnaphthylamine |
HEPES | 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid |
MTT | 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide |
DMSO | dimethyl sulfoxide |
AAC | Acoustic AC |
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Group | Peptide | Amino Acid Sequence a |
---|---|---|
Parent | V13K | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide |
Polar face group | K14D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide |
S11D/K14D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide | |
K14D/T15D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide | |
S11D/K14D/T15D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide | |
Non-polar face group | A12D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide |
F9D/A12D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide | |
A12D/V16D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide | |
F9D/A12D/V16D | Ac-K-W-K-S-F-L-K-T-F-K-S-A-K-K-T-V-L-H-T-A-L-K-A-I-S-S-amide |
Peptides a | tR (min) b | Benign c | 50% TFE d | ||
---|---|---|---|---|---|
25 °C | [θ]222 | α-Helix (%) e | [θ]222 | α-Helix (%) e | |
V13K | 44.48 | −5617.46 | 11.1% | −50,520.3 | 100% |
K14D | 41.93 | −2223.13 | 4.4% | −38,897.2 | 77.0% |
S11D/K14D | 40.77 | −3524.85 | 7.0% | −25,931.3 | 51.3% |
K14D/T15D | 40.41 | −1862.4 | 3.7% | −28,551.5 | 56.5% |
S11D/K14D/T15D | 39.59 | −2845.85 | 5.6% | −19,537.5 | 38.7% |
A12D | 41.46 | −4130.9 | 8.2% | −35,785.1 | 70.8% |
F9D/A12D | 40.04 | −2319.89 | 4.6% | −19,328.6 | 38.3% |
A12D/V16D | 39.79 | −3435.72 | 6.8% | −23,452.9 | 46.4% |
F9D/A12D/V16D | 39.06 | −1907.83 | 3.8% | −18,393.1 | 36.4% |
Peptides | MHC a (μM) | G+ MIC b (μM) | G− MIC b (μM) | ||
---|---|---|---|---|---|
S. aureus | S. epidermidis | P. aeruginosa | E. coli | ||
ATCC25923 | ATCC12228 | ATCC27853 | ATCC25922 | ||
V13K | 250 | 32 | 4 | 8 | 8 |
K14D | 125 | 32 | 16 | 16 | 32 |
S11D/K14D | 125 | 64 | 16 | 32 | 64 |
K14D/T15D | 250 | 64 | 16 | 32 | 64 |
S11D/K14D/T15D | 250 | 64 | 16 | 125 | 125 |
A12D | 125 | 64 | 16 | 16 | 32 |
F9D/A12D | 250 | 125 | 64 | 32 | 125 |
A12D/V16D | >500 | 125 | 125 | 32 | 125 |
F9D/A12D/V16D | >500 | 125 | 125 | 125 | 125 |
Peptides | IC50 (μM) a | |||
---|---|---|---|---|
HeLa | MIA PaCa-2 | HPAC | BxPC-3 | |
V13K | 16 ± 0.5 | 24.8 ± 0.6 | 7.6 ± 4.0 | 12.5 ± 1.2 |
K14D | 15.6 ± 4.1 | 17.9 ± 2.3 | 7.2 ± 5.2 | 20.0 ± 0.8 |
S11D/K14D | 30.6 ± 0.5 | 41.3 ± 2.6 | 25.5 ± 0.5 | 22.4 ± 2.3 |
K14D/T15D | 31.6 ± 2.1 | 47.1 ± 5.3 | 22.5 ± 0.5 | 24.1 ± 5.5 |
S11D/K14D/T15D | 41.1 ± 0.5 | 56.8 ± 3.3 | 54.2 ± 1.2 | 45.5 ± 5.6 |
A12D | 28.8 ± 0.8 | 29.9 ± 4.8 | 15.5 ± 1.7 | 45.1 ± 1.0 |
F9D/A12D | 59.8 ± 0.5 | 38.8 ± 2.9 | 42.4 ± 3.0 | 125.0 ± 3.7 |
A12D/V16D | 60.1 ± 1.2 | 46.2 ± 3.0 | 41.4 ± 1.4 | 125.0 ± 5.9 |
F9D/A12D/V16D | 125.0 ± 3.4 | 57.9 ± 2.8 | 95.6 ± 1.8 | 125.0 ± 1.8 |
Peptides | HEPES (nm) | PC/PG | PC/Chol | PC/SM/PE/PS/Chol | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Wavelength (nm) | Blue Shift (nm) | Intensity | Wavelength (nm) | Blue Shift (nm) | Intensity | Wavelength (nm) | Blue Shift (nm) | Intensity | ||
V13K | 353 | 329 | 24 | 459 | 344 | 9 | 486 | 330 | 23 | 582 |
K14D | 349 | 332 | 17 | 400 | 349 | 0 | 459 | 329 | 20 | 507 |
S11D/K14D | 347 | 332 | 15 | 346 | 345 | 2 | 414 | 331 | 16 | 363 |
K14D/T15D | 349 | 336 | 13 | 348 | 349 | 0 | 412 | 337 | 12 | 346 |
S11D/K14D/T15D | 345 | 339 | 6 | 324 | 343 | 2 | 308 | 335 | 10 | 345 |
A12D | 351 | 328 | 23 | 386 | 345 | 6 | 464 | 327 | 24 | 413 |
F9D/A12D | 350 | 335 | 15 | 339 | 348 | 2 | 456 | 331 | 19 | 338 |
A12D/V16D | 350 | 336 | 14 | 329 | 348 | 2 | 330 | 337 | 13 | 340 |
F9D/A12D/V16D | 346 | 335 | 11 | 300 | 346 | 0 | 261 | 337 | 9 | 316 |
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Sun, S.; Zhao, G.; Huang, Y.; Cai, M.; Yan, Q.; Wang, H.; Chen, Y. Enantiomeric Effect of d-Amino Acid Substitution on the Mechanism of Action of α-Helical Membrane-Active Peptides. Int. J. Mol. Sci. 2018, 19, 67. https://doi.org/10.3390/ijms19010067
Sun S, Zhao G, Huang Y, Cai M, Yan Q, Wang H, Chen Y. Enantiomeric Effect of d-Amino Acid Substitution on the Mechanism of Action of α-Helical Membrane-Active Peptides. International Journal of Molecular Sciences. 2018; 19(1):67. https://doi.org/10.3390/ijms19010067
Chicago/Turabian StyleSun, Shiyu, Guangxu Zhao, Yibing Huang, Mingjun Cai, Qiuyan Yan, Hongda Wang, and Yuxin Chen. 2018. "Enantiomeric Effect of d-Amino Acid Substitution on the Mechanism of Action of α-Helical Membrane-Active Peptides" International Journal of Molecular Sciences 19, no. 1: 67. https://doi.org/10.3390/ijms19010067