An engineered third electrostatic constriction of aerolysin to manipulate heterogeneously charged peptide transport

Abstract

Reading the primary sequence directly using nanopore remains challenging due to the complex building blocks of 20 proteinogenic amino acids and the corresponding sophisticated structures. Compared to the uniformly negatively...

β-Barrel Nanopores with an Acidic–Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

“… 32 , 33 In a recent communication, the aerolysin nanopore was engineered to detect negative and weakly positive peptides at near-physiological pH values. 34 …”

“…In one example, the differences among almost all 20 amino acids in polyarginine peptides were observed . Aerolysin mutants were shown to detect a range of model negatively charged peptides as well as phosphorylation and acetylation on τ-peptides. , In a recent communication, the aerolysin nanopore was engineered to detect negative and weakly positive peptides at near-physiological pH values …”

“…32,33 In a recent communication, the aerolysin nanopore was engineered to detect negative and weakly positive peptides at near-physiological pH values. 34 Here, we assessed the ability of two β-barrel nanopores, aerolysin and cytotoxin K (CytK), to capture a mixture of peptides originating from the protease digestion of lysozyme. We showed that in 1 M KCl solutions the wild-type nanopores could not resolve peptide mixtures at physiological or acidic pH values.…”

Nanopore-based technologies beyond DNA sequencing

“…Although the interactions between peptides and nanopores are likely to play an important role for a given class of nanopore 26 , other important properties of peptide, such as hydrophobicity, charge or folds, should be revealed instead. Another considerable step in this field of research was the realization that by lowering the pH to less than 4, most peptides can be nearly uniformly charged and captured irrespective of their chemical composition 61 , although an electro-osmotic flow was manipulated to capture peptides with a different net charge at near-physiological pH 62 . Based on peptide volume recognition, a single-molecule protein identifier has been proposed in which a protease is placed directly above a biological nanopore, and the fragmented peptides are sequentially read by a nanopore sensor (Fig.…”

A Comprehensive Single-molecule Experiment of Peptide Phosphorylation Detection with Biological Nanopores

“…在当今的后基因组时代，纳米孔道电化学领域的研究热点逐渐发展为单分子多肽/蛋白质的检测 和测序，磷酸化、乙酰化、甲基化等蛋白质翻译后修饰是其重要研究内容。笔者在制定实验方案时， 将兴趣为导向、学生为中心作为指导思想，综合考虑了实验的可操作性、创新性以及学生的适应性 和接受度，确保实验具有适度的挑战性，能够增强学生的学习信心和科研热情，充分发挥"科教融 合"对教学改革和高素质人才培养的重要促进作用。本实验利用突变型T232K/K238Q Aerolysin生物 纳米孔道检测多肽磷酸化，研究对象为丝氨酸磷酸化的Tau多肽，其与阿尔兹海默症等多种退行性疾 病相关。由于磷酸化增加了丝氨酸的负电性并改变了其体积，突变型T232K/K238Q Aerolysin纳米孔 道232位点正电荷的增加可增强静电相互作用，而238位点谷氨酰胺的引入则增加了分子过孔能垒， 因此，能够很好地应对因多肽过孔速度太快而难以实现准确测量的难题。本实验原理简单、现象明 确，便于学生理解和掌握，具有较强的可设计性和推广价值，不局限于多肽磷酸化检测，允许拓展 DNA损伤识别、蛋白构象研究、生物标志物检测、离子选择性测量等内容 [41][42][43][44][45][46][47] 。此外，MinION、 Orbit Mini、eONE等商品化仪器设备以及α-Hemolysin等商业化孔道蛋白也可应用于纳米孔道实验教 学，不同院校可根据具体情况选择合适的教学方案。在前期教学过程中，很多学生表示这是第一次 接触单分子实验，希望以后有机会能够参与相关课题研究，进一步了解纳米孔道电化学及其交叉领 域前沿进展。这充分表明了将具有重大应用前景的科学技术转化为本科教学内容，实现实验教学与 前沿研究并轨，对于培养学生的综合创新能力和推动"全面的化学教育"具有重要教学意义。 致谢：感谢研究生牛红艳和霍明珠的助教工作、张琳琳对纳米孔道仪器的调试和维护，以及本科学生刘宇航、储文 浩参与突变型生物纳米孔道的实验。 参 考 文 献…”