Studies On The Synthesis Of Transmembrane Peptides And Mechanism Of Transmembrane Domain Interactions

In this thesis, comparing the sequence of the two membrane proteins in various species through the NCBI protein database, we can obtain the highly conserved transmembrane domain of E-cadherin and the sixth transmembrane domain of β2AR respectively. We first used TOXCAT assay to determine dimerization of the transmembrane domain, then indentified the residues on the dimerization interface by site-directed mutagenesis. Then we used Fomc solid phase chemical synthesis to get the two transmembrane peptides, CD showed the second structure of peptide is typical α-helical. We also used SDS-PAGE to study the interaction of transmembrane peptide. In addition, in order to rich our experimental data we have conducted computational modeling to observe the dimerization of transmembrane domain.We found that CAT activity of transmembrane domain could been influenced by the length of inserted transmembrane domain. Mutating the leucine zipper motif and GxxxG motif in E-cadherin transmembrane domain revealed that leucine-zipper motif mediated dimerization not by GxxxG motif. Our computer molding results are consistent with the above experimental results. Based on TOXCAT results of β2AR, we found that CAT activity was sensitive to the length of inserted transmembrane domain to some extent. In fact the optimal length for the transmembrane domain in TOXCAT assay is not the longested one. Mutants assay revealed that the GxxxG motif could play a part in dimerization. Computer molding results also confirmed tha the GxxxG motif in the sixth transmembrane domain didn't mediate dimerization predominantly.In this thesis, the results provide a better understanding for relationship between structure and function of transmembrane protein.