Development Of The Ion Channel Polypeptide With PH Controllability

pH Low Insertion Peptides(pHLIPs)is a novel class of moderately polar membrane peptidesbased on pH-sensitive,whichinserts into the bilayer or cellmembraneas a transbilayer ?-helix.The insertion reaction proceeds rapidly and is fully reversible.pHLIPs has a dual delivery capability andtargetscells located in the acidic environmentat many diseased tissues,including the diagnosis and treatment of disease.M2 proton channel is a kind of pH-dependent selective proton channelin Influenza A virus lipid bilayer membranes and playsimportant role in viral infection and replication.The four?-helix transmembrane peptides of the M2 protein form a hydrophilic proton channel and the physical and chemical properties of the amino acid residues at the critical sites of the M2 protain transmembrane domain(M2TM)channel surface directly affect the diameter and hydrophilicity of the channel.According to the pH sensitive peptides and influenza virus Atransmembrane protein domains,we designed,synthesized,purificated series of polypeptides,found out the ion channel polypeptide with pH controllability by detection,it issoluble inwateron neutral condition,then spontaneously embedded in lipid bilayers,forming a-helix and cation channels after acidication.The experiments used solid phase peptide synthesis(SPPS)to synthetise peptide sequences,matrix assisted laser desorption ionization time-of-flight mass(MALDITOF-MS)to determine the target peptide,and reversed-phase high performanceliquid chromatography(RP-HPLC)to purify the Q series of peptides,and then freeze-dried.Firstly,we used fluorescence spectrophotometer to detect the fluorescence intensity changes of tryptophan located in the peptides andthe fluorescence peak corresponds to the left shift of the wavelengthin different environmentto find out the acid sensitive peptide sequences by they insert into a lipid bilayer.Then,the N terminal ofpeptide sequence reacted with 4-chloro-7-nitrobenzofurazan(NBD-Cl)by nucleophilic substitution reaction and NBD-dithionite quenching experiment toprobe the topology of the petide insertion.Acrodding to the experiments,we found that three peptide sequences:AC-Q10,AC-Q11,AC-Q12 have good acid sensitivity and the effect of pH on the fluorescence intensity of AC-Q10 D and AC-Q10 T showed that the peptides have good pH controllability.We determined the term into the membranewasCterminal of peptides.All these work had made full preparations for subsequent ion channel formation and ion channel activity experiments.