Size-controllable Assembly Of Transmembrane ?-Helical Peptide Nanochannels Using Molecular Templates

Transmembrane channels are the basis of molecular transport,neural signal transduction,and cell electrophysiological properties.Transmembrane channels have a wide variety of precise structures,and are currently applied in the fields of action potentials,transmembrane transport of materials,and nanopore single-molecule technology etc.Nevertheless,the pore size and the number of monomers/subunits in transmembrane channels are fixed and sole.Moreover,the formation mechanism of transmembrane channel is very complicated,especially as?-helical transmembrane channels usually have multiple transmembrane domains,making it difficult to control the channel size of transmembrane channels.In addition,the overall molecular weight of transmembrane channels is large,and the number of amino acids is at least 1,000.Such numerous amino acids disable the de novo design of transmembrane channel,and thus it is a challenge to construct them by total synthesis.Based on this,this thesis proposes a new strategy to assemble transmembrane channels and regulate channel size based on cyclodextrins as templates and?-helical peptides.We have modified naturally existed,cyclic and rigid molecules?-,?-and?-cyclodextrin to construct molecular assembly templates,designed and obtained new?-helical transmembrane peptides with high purity,and assembled the transmembrane peptides by click chemistry.The stable and functional transmembrane channels have been obtained by total synthesis and the size regulation of the transmembrane channel has been achieved.Detailed contents are summarized as follows:1.Research ReviewThe concept,structural characteristics and classification of transmembrane proteins are briefly introduced.The structures,functions and applications of large-sized?-helical ion channels,?-perforin and small-sized transmembrane peptides are reviewed.The current bottlenecks in the research of transmembrane channels,proposed strategies and innovations for this study have been given.2.Templates Synthesis for Assembly of Transmembrane ChannelUsing iodine and triphenylphosphine as electrophilic reagents,the hydroxyl group at primary side of cyclodextrins can be completely substituted to obtain per-6-deoxy-6-iodo-cyclodextrin.Further reaction with sodium azide can produce per-6-deoxy-6-azido-cyclodextrin.In addition,the iodide can be directly substituted by propargylamine to obtain cyclodextrins with a terminal alkynyl group.The products were verified by nuclear magnetic resonance(NMR),infrared spectrum(IR)and high-resolution mass spectrometry(MS).These cyclodextrin derivatives with terminal alkynyl or azide groups can be used in click chemistry to assemble various functional molecules at the primary side of cyclodextrin.3.Construction of Cyclodextrin-peptide AssemblyBased on the structural features and insertion mechanism of a known channel protein Wza,we redesigned a transmembrane?-helical peptide,lWza,containing a linker part and an alkynyl or azide group at N-terminus for assembly reaction.The transmembrane?-helical peptide 1Wza was assembled to the primary side of cyclodextrins by copper-catalyzed cycloaddition(CuAAC)reaction.Three assemblies(lWza)_n·CD(n=6,7 and 8)were obtained after separation and purification,and characterized by SDS polyacrylamide gel electrophoresis(SDS-PAGE),infrared spectroscopy,and MALDI-TOF mass spectrometry.This method is simple,effective,and may not destroy the stability of peptides,providing a new idea for the assembly of transmembrane proteins.4.Single-channel Analysis of Cyclodextrin-supported Transmembrane ChannelsBased on cyclodextrin templated?-helical transmembrane peptide assembly(lWza)_n·CD(n=6,7 and 8),a series of novel transmembrane channels have been constructed on the lipid membrane by planar lipid bilayer technique.Through single-channel analysis,the channel stability,pore size,current characteristics,and gating effects have been explored.It was found that macromolecule like chitosan can block and close the transmembrane channels transiently.In addition,the cyclodextrin-supported transmembrane channels possess the ability to interact with drug molecules by host-guest interaction.Cyclodextrin-supported transmembrane channels have structure stability and specific functions,thus laying a foundation for their application in nanopore single-molecule technique or as drug carriers.5.Size Regulation of Transmembrane ChannelIn order to make synthetic transmembrane channels more similar to natural channel proteins and achieve channel size regulation,this study has hydrolyzed the cyclodextrin templates in(lWza)_n·CD(n=6,7 and 8).After screening for amylase,we have found that maltose?-amylase can partially hydrolyze the cyclodextrin template,removing the rigidity of the cyclodextrin,and detemplated transmembrane peptide assembly(lWza)_n(n=6,7and 8)have been constructed.The hydrolyzed assembly can form stable open channels(at positive voltage)on the lipid membrane,and has a voltage-gating response under negative voltage.In this study,the stability mechanism of transmembrane channels(lWza)_n(n=6,7and 8)has been explained by molecular simulation,and accurate molecular models for each channels have been constructed,thereby realizing the size regulation of transmembrane channels.