Preliminary Study About Construction Of An Artificial Signal Transduction System On Bilayer Vesicles

The study used the signal transduction system of intracellular,which got the extracellular signal from the acceptor histine protein kinase AgrC of Staphylococcus aureus,as the imitated prototype.The polypeptide synthesized following the transmembrane region was incorparated in the artificial bilayer membranes vesicles under specific conditions,and the primary signal transduction system used to study the quorum sensing signal transduction mechanism of S. aureus was constructed.First of all,the amino acid sequence homology between different agr-type S.aureus was analyzed by DNAStar software.The analysis result shows that AgrC is acceptor histine protein kinase,and contains an N-terminal transmembrane region and an intracellular histidine protein kinase domain,which can be activated or inhibited by homologous or non-homologous AIPs. The transmembrane structure of different agr-type S.aureus was analyzed by TMHMM and TMpred software,and the target site between the acceptor(AgrC) and the donor(ALP) was determined.This study provides a theoretical basis for the construction of artificial membrane system and the further study of activation and inhibition of AgrC protein.Secondly,two cationic peptide lipids N,N-dihexadecyl-N^α[6-(trimethylammonio) hexanoyl]alaninamide bromide(N⁺C₅Ala2C₁₆) were synthesized from 1-hexadecylamine and 1-bromohexadecane through five steps including substitution and condensation reactions, which prepared material for artificial membrane synthetic.For the problem of the complexity of separation and purification process and the low yield,an improvement was made on the synthesis process and especially on the product purification method.The new synthesis method is simple,efficient,and lipid-peptide synthesis scale won't restricted by the purification methods and the total synthetic yield is up from 4.75%to 23%.At last,after analysis,an amino acid sequence,which has the closest relation to signal transduction among the six transmembrane regions of agr I S.aureus ATCC 6538,was designed,synthesized and modified.And then this polypeptide sequence was embedded to the artificial membrane using ultrasound method.The analysis results of the transmission electron microscopy and atomic force microscope show that vesicles and the embedded system are almost spherical and the diameter ranges from 50 to 200nm.The effect of environmental factors,such as pH,temperature and concentration of the system,on the stability of the embedded system was studied.And the results show that its strongest stability occurs at pH 7.0,30℃,and the molar ratio of peptide to N⁺C₅Ala2C₁₆ is 1:100.At the same time,the results of the characterization of the mosaic model by Circular dichroism and differential scanning calorimetry analysis indicate that the polypeptide can embed to the bilayer membranes vesicles stably and initiatively to form a embedded system.