pH敏感的抗菌肽LAH4与膜相互作用的电子顺磁共振研究

doi:10.5246/jcps.2019.11.071

中国药学(英文版) ›› 2019, Vol. 28 ›› Issue (11): 749-759.DOI: 10.5246/jcps.2019.11.071

• 【研究论文】 • 下一篇

pH敏感的抗菌肽LAH4与膜相互作用的电子顺磁共振研究

盛伊娜¹, 王申林², 刘国全^1*

1. 北京大学医学部药学院天然药物及仿生药物国家重点实验室, 北京 100191
2. 北京大学化学与分子工程学院, 北京 100871

收稿日期:2019-06-13 修回日期:2019-08-24 出版日期:2019-12-01 发布日期:2019-09-19
作者简介:Tel: +86-10-82801517, E-mail: guoquanliu@bjmu.edu.cn
基金资助:
Seed Fund of Peking University (Grant No. BMU2018MC003), Peking University Health Science Center (Grant No. BMU20160566), and the National Key Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2016YFA0501203) and the National Natural Science Foundation of China (Grant No. 31470727, 21874004).

Membrane binding of the pH sensitive antimicrobial peptide LAH4 studied by EPR spectroscopy

Yina Sheng¹, Shenlin Wang², Guoquan Liu^1*

1. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
2. College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

Received:2019-06-13 Revised:2019-08-24 Online:2019-12-01 Published:2019-09-19
About author:Tel: +86-10-82801517, E-mail: guoquanliu@bjmu.edu.cn
Supported by:
Seed Fund of Peking University (Grant No. BMU2018MC003), Peking University Health Science Center (Grant No. BMU20160566), and the National Key Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2016YFA0501203) and the National Natural Science Foundation of China (Grant No. 31470727, 21874004).

摘要/Abstract

摘要：

多肽与膜结合过程对于包括抗菌肽抗菌在内的多种生命过程都是非常重要的。我们利用基于电子顺磁共振技术的方法以pH敏感的多肽LAH4为研究对象, 在人工磷脂膜上研究了多肽与膜的相互作用。根据标记在多肽上的氮氧自由基谱图变化, 我们研究了LAH4与不同磷脂膜结合的亲和力及动力学信息。研究发现LAH4与带负电的POPG的结合明显强于DMPC及POPC。此外, pH同样影响LAH4与膜结合的速率, 酸性条件下明显增快的结合可能是LAH4酸性条件下抗菌活性增强的原因之一。本研究的结果和方法为后续在细胞上研究抗菌肽的与膜结合及穿膜过程提供了基础。

关键词: 电子顺磁共振, 抗菌肽, 多肽与膜相互作用

Abstract:

Peptide-membrane binding is vital for many biological events, including the bacteria combating by antimicrobial peptides. Using the pH sensitive LAH4 peptide as model, we employed a convenient electron paramagnetic resonance (EPR) method to study the peptide-membrane binding process in artificial phospholipid membranes. Based on spectral changes of the nitroxide radicallabeled to the peptides, we characterized binding kinetics and affinity of peptides to different phospholipid membranes. The binding affinity of LAH4 towards POPG was more than an order of magnitude higher than those towards DMPC and POPC. The binding kinetics showed that LAH4 initially bound to POPG much more quickly than to DMPC and POPC. Additionally, pH also affected the binding kinetics in LAH4-membrane interactions, which helped explain the pH dependent antimicrobial activity of LAH4. The method might be further used to monitor the membrane binding/cell penetration of antimicrobial peptide in living cells.

Key words: EPR, AMPs, Peptide-membrane interaction

中图分类号:

R965

Supporting:

Figure S1. The chromatograms of LAH4-26SL.

Figure S2. The Q-TOF-Mass spectrogram of purified LAH4-26SL of [M+4H]⁴⁺(right) and [M+5H]⁵⁺ (left).

Figure S3. Room temperature X-band EPR spectrum of LAH4-26SL bound to different phospholipid vesicles. Peptide concentration is 100 μM. (a) DMPC, pH = 7.0 with buffer; (b) POPC: pH = 5.4 without buffer.

Figure S4. Dependence of the 1/R_b on P/L to calculate the distribution coefficient. Peptide concentration is 50 μM.

Figure S5. EPR spectra of LAH4-26SL mixing with lipid vesicles after 10s vortex. Peptide concentration is 100 μM.

Figure S6. (a) EPR spectra of LAH4-26SL mixing with DMPC vesicles in PBS at pH = 5.0 mixed without vortex. Peptide concentration is 100 μM. L/P=1000. “0 min” means the state of LAH4-26SL before mixed with DMPC vesicles. (b) Evolution of I_HF in DMPC vesicles in PBS at pH = 5.0 mixed without vortex. Peptide concentration is 100 μM. L/P=1000.

盛伊娜, 王申林, 刘国全. pH敏感的抗菌肽LAH4与膜相互作用的电子顺磁共振研究[J]. 中国药学(英文版), 2019, 28(11): 749-759.

Yina Sheng, Shenlin Wang, Guoquan Liu. Membrane binding of the pH sensitive antimicrobial peptide LAH4 studied by EPR spectroscopy[J]. Journal of Chinese Pharmaceutical Sciences, 2019, 28(11): 749-759.

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pH敏感的抗菌肽LAH4与膜相互作用的电子顺磁共振研究

Membrane binding of the pH sensitive antimicrobial peptide LAH4 studied by EPR spectroscopy

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