Studies On Methylation Modification Of Protein Arginine By Nanopore Single-molecule Sensor

Among the many known types of posttranslational modifications,posttranslational methylation is one of the simple functionalized modifications that regulates the functions of hundreds of proteins and affects the interactions of proteins-proteins and proteins-nucleic acids.Identification of protein methylation is important for understanding complex cellular processes and the pathogenesis of some diseases.Since the changes caused by methylation modification are difficult to detect and identify,studying methylation modification has always been an challenge in the field of protein post-translational modification.In recent years,analysis sensors based on nanopore single molecule technology have attracted wide attention with the advantages of high sensitivity,no labeling,high resolution and are suitable for analysis of components with small differences.This technique has been successfully applied to the identification of peptide phosphorylation and glycosylation modification,however,because of methylation has very little effect on peptides and proteins,the research on peptide methylation modification using this technique has not been reported.In this paper,a complex sensor consisting ofα-hemolysin nanopore and octakis-（6-amino-6-deoxy）-γ-cyclodextrin was used to identify the methylated peptide.The binding properties of para-sulfonatocalix[4]arene with methylated peptide was also investigated.Our results provide some reference value for the study of the pathogenesis of cancer and other diseases regulated by protein methylation modification.Besides,the use of nanopore sensors to explore the binding properties between biomolecules provides a new platform for the development of new disease treatment.The main research contents are included as follows:1.Recognition of methylation of arginine residues in peptide based onα-HL-（WT）₇-am₈γCD nanopore sensor.Methylation modification does not change the total charge of proteins or peptides,but only slightly affect their size and hydrophobicity.Therefore,the identification of methylation modification is a difficult problem in the field of post-translational modification.Nanopore single molecule technology is a high resolution analytical technique to identify components with small differences,which can obtain fingerprint information of biomolecules.In order to enhance the interaction between the detected molecules and the recognition system,a novel recognition receptor was constructed using the complex of octakis-（6-deoxy-6-amino）-γ-cyclodextrin（am₈γCD）andα-hemolysin（α-HL）in this study.Due to the complex fluid properties in nanopore and the various non-covalent interactions between analytes and nanopore,we can identify the methylation modification of arginine residues in peptide from the two dimensions of dwell time and blocking current.By varying the electrophoretic forces and electroosmosis of the analytes in the nanopore under different experimental conditions,we explored the optimal recognition conditions,analyzing and discussing the different interactions for methylated and unmethylated peptides with the recognition receptor.The results show that this recognition receptor has excellent performance in the recognition of methylation modification,providing effective recognition information from the single molecule level and developing a new research idea for the recognition and detection of protein post-translational modification.2.Investigation of the binding properties of methylated peptide with para-sulfonatocalix[4]arene based onα-HL-（WT）₇-am₈γCD nanopore sensor.Post-translational modification of proteins has great influence on the interaction of protein-protein,protein-nucleic acid and protein-organic molecules.At present,most studies have analyzed the different methylation modification types of lysine residues based on the strong binding ability,while there are few reports on the binding properties between methylated arginine residues and SC₄.In this study,the effect of single methylation modification of arginine residues on the binding of peptides to SC₄ was investigated based on the characteristic current signal generated.The results showed that peptide containing methylated arginine residues had stronger binding ability to SC₄,the concentration ratio of analytes,p H and ion strength of solution had an obvious influence on the binding ability.This method provides a new strategy for studying the regulatory mechanism of protein methylation modification on biological system at the single molecular level.