Development Of New Methods For Chemical Synthesis Of S-palmitoylated Membrane Proteins And New Strategy For Peptide Modification To Regulate The Function Of Membrane Protein

Protein chemical synthesis can manipulate the structure of peptide/protein on the atomic scale to obtain protein samples that are difficult to obtain by other methods,such as D-proteins,unnatural peptides/proteins,and site-specific post-translational modified proteins,which have been widely used in research fields such as biochemistry,biophysics and biomedicine.Currently,more and more water-soluble proteins have been prepared by chemical synthesis,such as post-translational modified nucleosomes,glycosylated erythropoietin(EPO),and mirror DNA polymerase.However,there are very few cases of membrane proteins,which is another important family of proteins.Membrane proteins are involved in many important physiological process,including signal transduction,molecular transport,enzyme catalysis,immune response,and apoptosis,etc.So they are important targets for drugs.However,membrane proteins have very hydrophobic properties which make them very difficult to synthesize.In order to obtain membrane proteins by chemical synthesis,scientists have developed a series of new methods and new strategies,including addition of solubilizing reagents,heating and modification of removable solubilizing tags,especially removable backbone modification(RBM)strategies.It is worth mentioning that the RBM strategy makes membrane peptides/proteins similar to water-soluble proteins by installing solubilizing tags on the backbone,which is convenient for separation,purification,ligattion and characterization of membrane protein,thus enabling the efficient preparation of small and medium membrane proteins.Despite these studies,there are still a series of problems to be solved in the chemical synthesis of membrane proteins,including:First,how to prepare more complex membrane proteins,such as S-palmitoylated membrane proteins?Secondly,how to efficiently prepare larger membrane proteins?Finally,how to obtain active molecules for highly specific targeting and regulation of membrane protein?In response to these problems,(1)We have developed a third-generation removable backbone modification strategy(RBMGABA)based on a novel self-cyclized phenolic hydroxyl protecting group(?-aminobutyric acid,GABA),which can efficiently prepare S-palmitoylated transmembrane peptides;Based on a new generation of RBMGABA-assisted Ser/Thr ligation(STL),we have achieved efficient ligation of natural S-palmitoylated transmembrane peptides.In addition,S-palmitoylated protein is a classic case that cannot be prepared using native chemical ligation(NCL),so this work also proved the necessity of developing non-NCL methods for the first time.(2)We developed a new combination ligation method of N-to-C sequential NCL and STL.This strategy is not a simple combination of STL and NCL.The key lies in the development of the peptide salicylaldehyde S,S-propanedithioacetal(SALPDT)-ester prepared by a new 1,3-propanedithiol-mediated reaction,which is compatible with NCL.After the NCL is completed,the structure can also be activated by N-chlorosuccinimide/silver nitrate(NCS/AgNO3)into SAL-ester for the subsequent STL.The NCL-STL strategy combines the advantages of highly efficient peptide hydrazide-based NCL and S-palmitoylation compatible STL,which ensures the smooth preparation of S-palmitoylated IFITM3 containing 133 amino acids.The research also illustrates the importance of developing new chemistry to solve difficulties in protein chemical synthesis.(3)The strategy of replacing non-reducible thioether bond with disulfide bond will be a more universal and effective detection method for disulfide bond exchange between disulfide bond-rich peptides and their receptors.The key to this strategy is the use of diamino diacids containing thioether bond in SPPS.This method allows site-specific incorporation of non-reducible thioether bonds into Hepcidin.The functional study of these Hepcidin analogs shows that the interaction between Hepcidin and Ferroportin does not require intermolecular disulfide bond exchange.This research points out the direction for the subsequent drug development and provides a more effective solution for the study of the interaction between other disulfide-rich peptides and receptors.