The Study Of The Interaction Between Cyclodextrin Adapters And Protein Nanopore

In recent years,the technology of single molecule detection with excellent sensitivity and selectivity aroused extensive attention.Biological nanopore based on a-hemolysin(a-HL)has been widely used to stochastic sense of various analytes,such as metal ions,organic molecules,peptides,DNA/RNA and proteins.Stochastic detection is an approach to sensing that depends on analytes modulating the ionic current flowing through the pore,analyzing characteristic signature(residence time and amplitude)to realize qualitative and quantitative detection.However,if the molecular size of analyte is too small,it is hardly to detect the ionic current fluctuation within the a-HL nanopore.A different strategy was discovered,which utilized noncovalently attached molecular adapters,to increase the recognition of the small analytes.Due to the dimension of cyclodextrin matching with the a-HL nanochannel,it is used to be an adapter to lodge in the lumen of the the α-HL pore,the subsequent binding of analyte molecules causes a reduction in current flowing through the pore permitting the analyte to be identified and quantified.Here,we focus on the analysis of heptakis-(6-deoxy-6-amino)-β-cyclodextrin(am7βCD)with the protein nanopore to screen out the mutant protein with strong affinity for cyclodextrin.In addition,two cyclodextrin molecules with opposite charge were simultaneously lodged in the lumen of pore fore the first time,which formed a unique cavity.The cavity is important for further testing of small molecules.The important conclusions of this work are summarized as follows:1.The interaction of a single cyclodextrin with protein nanoporeThe experiments were carried out to study the interaction between am7βCD molecule and protein pore from three aspects.Firstly,we designed three mutant proteins,(K147N/M113F)7、(M113F)7 and(K8A/M113R)7,to study the effect of the mutant sites and residues for the interactions of them.The research showed that the the amino acids of the side chains at position 113 play a key role to the interaction of them,and mutated phenylalanine possessed a strong affinity for cyclodextrin.Compared(K147N/M113F)7 with(M113F)7,mutated to lysine at position 147 increased the entry frequency of am7βCD molecule.Secondly,studying the interaction of am7βCD molecule and(K147N/M113F)7 mutant pore varies with the transmembrane voltages.The results showed that the addition of am7βCD to the trans compartment produced reversible partial blockades of the ionic current.By contrast,no current block was detected when am7βCD was added from the cis side.And higher voltage would result in an increase in the residence time of amyβCD molecule within the lumen of the protein pore.Lastly,the relationship of the interaction of them and the pH of the electrolytes was investigated.The results demonstrated that am7βCD and protein pore with high affinity along with the increase of pH of the electrolyte.2.Assembled two cyclodextrins into the protein nanoporeHere,we prepared WT7,(K147N/M113F)7 and(K8A/M113R)7,three different proteins.Heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin heptasodium salt(HDAS-βCD)with negative charge and am7βCD molecule with positive charge were respectively added to the two sides of bilayer,cis and trans compartments,to select one simultaneously to interact with two cyclodextrins under applied positive potential.Our studies demonstrated that WT7 and(K147N/M113F)7 protein pores only could bind with am7βCD in the trans side of the bilayer,while the two cyclodextrins could be simultaneously assembled into the lumen of the(K8A/M113R)-7 protein pore.Then,it resulted in the extent of ionic current blockage increased.In addition,we detected the interaction of two cyclodextrins and protein pore at different transmembrane voltages and and the pH of the electrolyte.The results showed that residence time of the two cyclodextrins within the lumen of nanopore increased with an increase in transmembrane voltage.The responsible time is 36.48 ms at+160 mV,which is the seven times of+100 mV.However,the dwell time of the cyclodextrins decreased with an increase in the pH of the electrolyte.The corresponding event responsible time at pH 5.5 and pH 11.5 were 90.47 ms and 0.76 ms,respectively.By optimizing the transmembrane voltage and pH of electrolyte,the suitable degree of current blockage and long residence time of two cyclodextrins blockage could be obtained.Assembled two cyclodextrin molecules with opposite charge simultaneously into the lumen of α-hemolysin pore formed a unique cavity,which is important for further testing of small molecules.