Study The Trypsin Catalytic Kinetics And The Interaction Based On Nanopore Sensor

Protein is the cellular component that maintains its biological structure and regulates homeostasis.It is important for the study of the activity and structure of proteins to understand their biological functions and disease diagnosis.Because proteins are in eternal motion,they lead to complex conformational states,kinetics,interactions,and temporal spatial distribution.In recent years,nanopore single-molecule technology has received extensive attention due to its advantages of label-free,high throughput selectivity.By monitoring the ion current modulation caused by the molecules to detect chemical reaction and individual molecules,this technology will also become an important way to diagnose in the future.Nanopore single-molecule technology has been widely used in the analysis of DNA,small organic molecules,peptides,proteins,etc.However,due to the complex structure of the protein,and their diameter is usually larger than the size of the nanopore.Thus,there are still some challenges that the direct use of nanopore single-molecule technology to explore proteins.This paper has studied the process of trypsin hydrolysis and the interaction mechanism between trypsin molecular with nanopore.We used polyamine-modified?-cyclodextrin as a recognition element for?-hemolysin(?-HL)nanopores to real-time monitoring of the process of trypsin hydrolysis of small organic molecules and analysis of the kinetics of the process.In addition,the interaction between trypsin and nanopore is directly detected through the nanopore,and single-molecule analysis of protein-nanopore interaction is realized.The main research contents of this paper are as follows:1.Using polyamine-substituted cyclodextrin as the recognition component for real-time monitoring of trypsin catalyzed BAEE process.A mutant?-hemolysin(M113R)₇ protein nanopore equipped with polyamine decorated?-cyclodextrin(am₇?-CD)is employed as a sensing platform to real-time monitoring trypsin enzymatic reaction.We have quantitatively analyzed the event frequency of the catalytic product N?-benzoyl-L-arginine(BA)with time and plotted the Lineweaver-Burk curve to calculate kinetic parameters.The calculated kinetic parameters were consistent with the conventional spectroscopy method,indicating the feasibility of the method for detecting enzyme kinetics.Meanwhile,the effects of temperature,metal ions on trypsin catalytic activity were investigated.Calcium ions can form a stable complex with trypsin which may increase the autolysis stability and be beneficial to the reactive activity.Proper elevated temperature was conducive to the occurrence of enzymatic reaction.Significantly,this sensing strategy eliminates the need for complex synthetic substrate processes and can only cause the current modulation of the product,preventing interference from the enzymatic cleavage substrate,thus the entire detecting enzymatic processes are more simplified.This method should be reliably used for other types of enzyme activity detection in nanopore analysis.2.The nanopore sensor for detecting the interaction between trypsin and nanopore at the single molecule level.Protein translocation across biological membranes process involves protein-protein and protein-pore interactions.The study of the protein-pore interaction at single-molecule resolution is still missing due to the considerable complexity in the translocation machineries and the heterogeneity of charges on the protein surface.Here,we used high-resolution nanopore single-molecule technology to directly explore the interaction between trypsin and?-hemolysin nanopore.The interactions were explored by changing the mutation site of?-hemolysin,altering voltage,pH experimental conditions,revealing the balance of the electroosmotic flow,electrophoretic force and electrostatic interaction of driving proteins into the nanopore.The sensor provides a new way to label-free and easily study protein-nanopore interactions.